Macrophage tumor necrosis factor-alpha release is induced by contact with some tumors.

The purpose of this study was to determine whether macrophages were directly stimulated by tumor cells to release TNF-alpha. We found that several murine and human tumor cell lines and crude cell membrane vesicles prepared from these tumor cells stimulated pyran copolymer-elicited murine peritoneal macrophages (PEM) to release as much as 362 +/- 69 (mean +/- SE) units of TNF activity per 10(6) PEM in vitro. By contrast, several nontransformed cells, including Con A-stimulated splenic leukocytes and CTLL cloned T lymphocytes, failed to stimulate PEM to release TNF. Antibody and complement-mediated depletion of macrophages abrogated the release of TNF; whereas depletion of NK cells and T lymphocytes did not affect tumor-stimulated TNF release, suggesting that tumor cells directly stimulated PEM to release TNF. Tumor-stimulated TNF release was rapid, peaking in 2 to 3 h with subsequent loss of TNF activity from the medium. In the absence of tumor, PEM contained detectable levels of TNF mRNA, but did not release functionally active TNF. The addition of P815 tumor cell membrane vesicles increased both TNF mRNA levels, peaking at 1 to 2 h, and release of high levels of TNF activity. Confounding effects of endotoxin were excluded by the resistance of tumor-stimulated TNF release to neutralization by polymixin B, and by the equivalent responsiveness of PEM from endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive (C3H/HeN) mice to stimulation by tumor cells. Factors which stimulated PEM to release TNF could be extracted from tumor cell membrane, with 77% of the macrophage-stimulating activity recoverable in aqueous phase. In conclusion, we have demonstrated that some tumor cell lines express specific characteristics which can be recognized by macrophages and which stimulate macrophages to release TNF.

Immortalized retinal neurons derived from SV40 T-antigen-induced tumors in transgenic mice.

Immortalized retinal neurons have been established in tissue culture from retinal tumors arising in transgenic mice. The mice carry the SV40 T-antigen under the control of 5' flanking sequences from the human phenylethanolamine N-methyltransferase (PNMT) gene in order to target oncogene expression to adrenergic cell types. The retinal cultures contain a proliferation population of T-antigen-positive cells with a neuronal morphology that includes formation of extensive neuritic processes. We identified the cells as amacrine-derived neurons by immunofluorescence using the cell-specific monoclonal antibodies VC1.1 and HPC-1. The cells also express all three neurofilament subunits and GAP-43. These results indicate that CNS neurons can be transformed in transgenic animals to generate cultured cells with many properties of mature neurons.

A double quantum coherence transfer proton NMR spectroscopy technique for monitoring steady-state tumor lactic acid levels in vivo.

If proton nuclear magnetic resonance (1H NMR) spectroscopy is to provide a clinically useful modality for monitoring tumor growth and treatment, the technique must be able to unambiguously detect steady-state metabolite concentrations in human tumors and differentiate these from normal tissue levels. To address this problem, a two-dimensional double quantum coherence transfer spectroscopy (2DDQCT) method was developed and tested in a series of tumor cell lines implanted in mice. Lactate-edited proton NMR spectra were determined from a roughly 1-cm3 region of interest in EMT6, RIF-1, and fibroma. In two-dimensional data matrix representations of the 2DDQCT experiments (double quantum frequency on the vertical axis and chemical shift on the horizontal axis) the lactate signal (330 Hz with the transmitter set at the water resonance) was well-resolved from lipid (480 Hz, 600 Hz). The resolution in the double quantum dimension was also sufficient to conclude that a detectable level of alanine, which would reside at 358 Hz, was not present in the three tumor types. Following the NMR experiment, tumors were chemically assayed for lactate giving 8.17, 9.1, and 6.73 mumols/g wet wt for RIF-1, EMT6, and fibroma, respectively. This technique is likely to provide a noninvasive method for monitoring the steady-state lactic acid levels in small tumors before and after therapy, as well as in tissues with impaired oxygen delivery using clinical and research NMR systems.

Structure and function of laminin: anatomy of a multidomain glycoprotein.

Laminin is a large (900 kDa) mosaic protein composed of many distinct domains with different structures and functions. Globular and rodlike domains are arranged in an extended four-armed, cruciform shape that is well suited for mediating between distant sites on cells and other components of the extracellular matrix. The alpha-helical coiled-coil domain of the long arm is involved in the specific assembly of the three chains (A, B1, B2, and possible variants) of laminin and is the only domain composed of multiple chains. It is terminated by a large globular domain composed of five homologous subdomains formed by the COOH-terminal part of the A chain. Sites for receptor-mediated cell attachment and promotion of neurite outgrowth reside in the terminal region of the long arm. A second cell attachment site, a cell signaling site with mitogenic action, binding sites for the closely associated glycoprotein nidogen/entactin, and regions involved in calcium-dependent aggregation are localized in the short arms. These domains, which to a large extent are composed of Cys-rich repeats with limited homology to EGF, are the most highly conserved regions in laminins of different origin. At present, most structural and functional data have been collected for a laminin expressed by a mouse tumor, which can be readily isolated in native form and dissected into functional fragments by limited proteolysis. Increasing information on laminins from different species and tissues demonstrates considerable variations of structure. Isoforms of laminin assembled from different chains are focally and transiently expressed and may serve distinct functions at early stages of development even before being laid down as major components of basement membranes.

Image cytometry DNA analysis of diethylnitrosamine-induced dysplasias and invasive squamous cell carcinomas of the esophagus in mice.

The nuclear DNA content was assessed by image cytometry in squamous epithelial dysplasias and invasive squamous cell carcinomas of the esophagus induced by diethylnitrosamine (DEN) in mice. The study comprises 48 lesions: 27 lesions with low grade (i.e. slight and moderate) dysplasia, 18 with high grade dysplasia (i.e. severe dysplasia and CIS), and 3 with invasive squamous cell carcinoma. In addition, 5 parallel run control specimens were also investigated. The results demonstrated that only 3.7% (or 1/27) low grade dysplasias but as much as 72.2% (or 13/18) high grade dysplasias, and all three invasive squamous cell carcinomas displayed non-diploid DNA values. Three of 18 high grade dysplasias and all three invasive squamous cell carcinomas demonstrated aneuploid cell nuclei. The results of the present work indicate that the esophageal mucosa of the mouse permit investigation--under controlled conditions--of the nuclear DNA alterations occurring during carcinogenesis in this organ. The results presented herein thus substantiate the theory of increasing DNA aberrations occurring during carcinogenesis in the human esophagus.

Presence of a receptor for the active component of Iscador in ascites fluid of tumour bearing mice.

Tumour bearing mice exhibit a specific "receptor" in the ascites fluid which binds with the active component isolated from Iscador. This "receptor" was found to be a protein which inhibited the cytotoxicity of Iscador and its isolated active component at low concentration. The receptor protein was also found in the sonicates of tumour cells which are susceptible to the action of Iscador but not in lymphocytes which were not susceptible to Iscador or its isolated active component. The receptor was separated on a Sephadex G-50 column. Activity was lost upon heat denaturation and dialysis.

Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor.

We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation. A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium. Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week. This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month. We report here that the dedifferentiation of P15 occurs in two stages. One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by approximately 70% and remained at this reduced level for at least one month. After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D). Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN). Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day. There was also an increase in the amount of dopa-positive TGN within one day. Melanosomes first appeared after three days and became numerous after one week. Upon removal of fish serum, these re-induced cells (P15D1) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells. We also report that a protein designated as p75 (Mr approximately 75,000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.

Mouse heart laminin. Purification of the native protein and structural comparison with Engelbreth-Holm-Swarm tumor laminin.

Laminin was selectively extracted from different mouse tissues using EDTA-containing buffer. By immunoblotting with an antiserum raised against mouse Engelbreth-Holm-Swarm (EHS) tumor laminin, such extracts could be shown to contain laminin-like molecules with a low apparent proportion of A chain to B chains. Native laminin was purified from mouse heart tissue and was shown to have an aberrant polypeptide composition as compared to mouse EHS tumor laminin. Most prominently, mouse heart laminin contains an Mr 300,000 polypeptide which is not antigenically related to the A or the B chains. Furthermore, nonreducible polypeptide components were seen with apparent Mr values of 600,000 and 900,000. The Mr 600,000 component contains epitopes shared with both EHS tumor laminin and the Mr 300,000 polypeptide and possibly represents a covalently cross-linked complex of an A or B chain with the Mr 300,000 chain.

Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures.

Four malignant tumors induced in mouse by different experimental procedures were compared as regards their high-mobility-group (HMG) proteins. All tumors showed the complete set of three HMG proteins which we call HMGI-C, I-D, and I-E. The presence of the three HMGI proteins is a characteristic of the transformed phenotype regardless of whether the tumor was chemically, virally, or spontaneously derived. However, the level of expression of the HMGI proteins is not constant in the four tumors. Using reverse-phase HPLC, the individual HMGI proteins were isolated from the spontaneously derived tumor (Lewis lung carcinoma) and shown by amino acid analysis to be similar to those previously obtained from a tumor grown in nude mice by inoculation of in vitro-transformed cells.

Promotion by bombesin of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of bombesin on the incidence, number, histological type, and depth of involvement of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in male Wistar rats. Rats received alternate-day s.c. administration of 20 or 40 micrograms/kg body weight of bombesin in depot form after p.o. treatment with the carcinogen for 25 weeks. Prolonged administration of bombesin at 40 micrograms/kg led to a significant increase in the incidence and number per rat of gastric cancers of the glandular stomach at Week 52. In rats that had received alternate-day injections of 20 micrograms/kg of bombesin, the number of gastric cancers per rat, but not the incidence of cancer, was significantly more than in untreated rats. However, bombesin at both dosages did not affect the histological appearance of the lesions or their depth of involvement. At Weeks 30 and 52, norepinephrine concentrations in the fundic and antral portion of the gastric walls and labeling indices in the antral and fundic mucosae were significantly higher in rats treated with bombesin at both dosages than in untreated rats. These findings indicate that bombesin enhances gastric carcinogenesis after administration of N-methyl-N'-nitro-N-nitrosoguanidine is stopped and that this effect may be related to its effects in increasing tissue norepinephrine concentrations in the stomach wall and increasing cell proliferation in the gastric mucosa.

Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines.

Lung and liver tumors were induced in female A/J mice after treatment for 7 weeks (3 times/week, i.p.) with either 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (50 mg/kg) or nitrosodimethylamine (NDMA) (3 mg/kg). Both compounds can be activated via alpha-hydroxylation to methylating agents, while NNK may also undergo hydroxylation at the N-methyl carbon to form a pyridyloxobutylated adduct. The purpose of these studies was to identify and characterize the activated oncogenes present in tumors induced by NDMA and NNK. Following transfection of high molecular weight DNA onto NIH/3T3 mouse fibroblasts, transforming genes were detected in 90% of both NNK- (10 of 11) and NDMA- (9 of 10) induced lung tumors. In contrast, transformation of NIH/3T3 fibroblasts was observed only in 40% (2 of 5) and 13% (1 of 8) of the liver tumors from NNK- and NDMA-treated mice, respectively. Southern blot analysis indicated that the transforming gene present in all lung tumors was an activated K-ras oncogene. Both rearranged bands and amplified signals were detected in the transfectants. The one transformant from the NDMA-induced liver tumor contained an activated K-ras gene. In contrast, the two liver transformants from NNK-induced tumors did not contain an activated ras or raf gene. Hybridization with oligonucleotide probes that were centered around either codon 12 or 61 of the K-ras gene were utilized to localize the mutations. Activation of this gene appeared to occur largely via a mutation in codon 12 (15 of 20 transformants) and was observed with a similar frequency in pulmonary tumors induced by either compound. The remaining mutations were found in codon 61. The specific mutation within these two codons was determined by amplifying the exon containing the base change, followed by direct sequencing. With one exception the mutation observed in codon 12 was a GC to AT transition (GGT to GAT). One transformant contained a GC to TA transversion. The activating mutation detected in codon 61 was always an AT to GC transition of the middle A (CAA to CGA). The GC to AT mutation observed in codon 12 is consistent with the formation of the O6-methylguanine adduct. Similar concentrations (23 to 32 pmol/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibrinolytic inhibitors from the experimental rat epithelioma.

Guerin epithelioma, a highly metastatic tumour implanted to Wistar rats contains two inhibitors of fibrinolysis which can be detected with the use of zymographic techniques. The first one--with Mr about 48000 forms SDS-stable complex with urokinase. The second--with Mr about 7000 inhibits fibrinolytic and amidolytic activity of plasmin.

Ligand-induced phosphorylation of a murine tumor surface protein (TSP-180) associated with metastatic phenotype.

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.

Use of monochlorobimane for glutathione measurements in hamster and human tumor cell lines.

The use of monochlorobimane (MCIB) as a fluorescence label for glutathione (GSH) quantitation was investigated in human tumor cell lines. When MCIB was used with a hamster fibroblast cell line under conditions where GSH was either depleted or elevated, an excellent correlation between bimane-GSH fluorescence and the standard cyclic GSH reductase assay (Tietze's) was accomplished. When the MCIB technique was applied to a human lung adenocarcinoma cell line, little or no GSH labeling was noted even at MCIB levels 10X higher than that used for the hamster line. HPLC analysis suggested that the source of the problem may be the affinity for MCIB to glutathione S-transferase. By using higher dye concentrations and longer staining times, adequate staining was possible. While the MCIB technique may have problems quantitating GSH levels between cell types, the possibility of examining GSH heterogeneity in solid tumor biopsies remains feasible.

Demonstration of tumor-selective retention of fluorinated nitroimidazole probes by 19F magnetic resonance spectroscopy in vivo.

We have evaluated two fluorinated misonidazole analogues, Ro 07-0741 and CCI-103F, as potential probes for the non-invasive identification of hypoxic tumor cells by 19F magnetic resonance spectroscopy (MRS) in vivo. The equipment used was a 1.9 T Oxford Research Systems TMR-32 spectrometer, fitted with a 15 mm diameter surface coil. Signal was readily detectable, with similar intensity from EMT6 tumor, liver, and brain at early times (1-2 hr) after i.v. injection in BALB/c mice, indicative of an initial uniform biodistribution of parent probes. At later times (5-10 hr) there was a progressive reduction in signal intensity from brain and liver, but tumor levels remained constant or declined more slowly. This is illustrated by tumor/brain ratios at 6-7 hr of 2.9 (Ro 07-0741) and 4.2 (CCI-103F). In 4/5 mice analyzed at 20-24 hr after Ro 07-0741, and 1/2 following CCI-103F, tumor signal remained detectable. This occurred in the absence of parent probe as measured by HPLC, suggesting the involvement of a product of nitroreductive bioactivation. Studies with KHT and RIF-1 tumors in C3H/He mice showed a similar trend but retention in RIF-1 was less dramatic, and this was consistent with the known hypoxic fractions and comparative in vivo nitroreductase activities. These promising results support the continuing development of 19F nitroimidazole probes for non-invasive identification of hypoxic cells in vivo.

Laminin receptor expression on murine tumor cells: correlation with sensitivity to natural cell-mediated cytotoxicity.

Previous studies have identified a relationship between the presence of cell surface laminin receptors on murine tumor cells and sensitivity to killing by natural killer (NK) cells. On the basis of these observations, we suggested that laminin and laminin receptors may function to facilitate the interaction of NK-sensitive murine target cells with NK cells. Our original studies were conducted with a number of genetically unrelated tumor cell lines. In order to extend these earlier observations, studies have now been conducted in which sensitivity to NK-mediated lysis and responsiveness to laminin were compared in a number of variant populations derived from the NK-sensitive cell lines Yac-1 and RL-1 and from the NK/NC-resistant line P815. All of the lines which interacted with murine NK cells as indicated by sensitivity to killing and/or by ability to "cold-target" compete with the killing of sensitive Yac-1 cells were able to bind 125I-laminin and to respond to laminin in an aggregation assay. Of 4 NK-resistant populations identified in these studies, 3 failed to respond to laminin. These studies indicate that even among genetically related tumor cell lines there is a relationship between laminin receptor expression and interaction with NK cells.

Solubilization and characterization of pituitary thyrotropin-releasing hormone receptors.

Thyrotropin-releasing hormone (TRH) receptors were solubilized from a rat pituitary tumor cell line, GH4C1, with digitonin. Convenient assays were developed based on the ability of hydroxylapatite and polyethyleneimine-soaked glass fiber filters to adsorb the solubilized [3H]methyl-TRH-receptor complex but not free [3H]methyl-TRH. The kinetics of [3H]methyl-TRH binding to solubilized receptors were extremely temperature dependent. Binding reached equilibrium at 10-20 nM [3H]methyl-TRH in 30 min at 23 degrees and 6 hr at 0 degree. The half-times for dissociation were less than 5 min at 23 degrees and 7.6 hr at 0 degree. Equilibrium binding experiments yielded linear Scatchard plots at 0 degree with Kd = 3 nM, whereas the Kd was greater than 20 nM at 23 degrees. A series of TRH congeners displaced [3H]methyl-TRH with the rank order reported for membrane receptors, N3-methyl-HisTRH greater than or equal to TRH greater than pGlu-N3-methyl-HisProNH(CH2)6NH2 greater than or equal to pGluHisProNH(CH2)6NH2 greater than pGluHisTyrNH2 much greater than TRH free acid. The antagonist chlordiazepoxide exhibited an IC50 of 10 microM. [3H]methyl-TRH binding to solubilized receptors displayed a broad pH optimum, from 6.5 to 7.5. The solubilized receptor could be obtained from cultured GH4C1 cells and in much larger quantities from GH4C1 tumors. Tumors from 12 rats yielded greater than 700 pmol of specific soluble TRH binding activity (1 g of protein). The solubilized receptor could be purified 10-20-fold by chromatography on wheat germ agglutinin columns and could be concentrated by adsorption on either DEAE-Sephadex or hydroxylapatite. The procedures outlined allow the solubilization of pituitary TRH receptors from a rich and abundant source, the rapid and reproducible assay of [3H]methyl-TRH binding, and substantial enrichment of receptor activity. These findings should be valuable for the purification and identification of the TRH receptor protein.

[Ultrastructural studies of tumors transplanted into nude mice using the TTK-1 cell lines derived from normal human early decidual tissue].

Tumors transplanted into nude mice using the TTK-1 cell lines [TTK-1(E) and TTK-1(F)] derived from normal human early decidual tissue were studied morphologically. The epithelial-like cell line TTK-1(E) and the fibroblast-like cell line TTK-1(F) were maintained in culture through one hundred and ten subcultures since July 1979. Rapidly growing tumor nodules formed at the implantation sites. The incidence of tumor growth was 100% for both cell lines. Histologically the tumors were composed of poorly-differentiated cells arranged in a cord-like structure and showed typical malignant characteristics. Immunohistochemical studies, electron microscopy and immunocytochemical studies revealed that the tumors from the two cell lines differed in many respects. The tumors formed by TTK-1(E) showed epithelial characteristics and the tumors formed by TTK-1(F) showed both epithelial and mesenchymal characteristics. Therefore, TTK-1(E) might be useful as an in vitro model of endometrial cancer and TTK-1(F) as an in vitro model of both endometrial cancer and endometrial stromal tumor (containing mixed mesodermal tumor). These tumors will be valuable for future studies of the tumorigenicity and therapy of uterine malignant tumors. They may reflect the various functions of decidual tissue.

[Immunological detection of carcinogen-DNA adducts].

Antibodies raised in rabbits against DNA modified with the carcinogen 4-hydroxyaminoquinoline 1-oxide or benzo (a) pyrene have been used in combination with an avidin-biotin peroxidase complex staining method to locate DNA adducts in paraffin sections of mouse tissues. The specificity of immunostaining was confirmed in several ways. When various doses of carcinogen solution were injected s.c. into isolated portions of mouse skin clamped off with ring-shaped forceps, the immunohistochemical staining of carcinogen-DNA adducts in the nuclei of epithelial cells, fibroblasts and panniculus carnosus cells increased dose-dependently. Nuclear staining was absent in animals given injections of isotonic solution only, and the intensity of staining correlated well with the level of unscheduled DNA synthesis demonstrated autoradiographically. 4HAQO-DNA adducts were observed in all target organs of 4HAQO tumorigenesis (i.e., lung, trachea, pancreas, uterus, vagina, skin, and colon) after injection of the carcinogen. Nuclear staining was absent or low in nontarget organs, including the liver and brain. Considerable variation was found in staining levels between cell types and different anatomic locations of cells within each target organ. The intensity of immunohistochemical staining correlated well with the numbers of 4HAQO-DNA adducts measured by the radio-labeling technique.