Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

Glomerular basement membrane proteoglycans are derived from a large precursor.

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.

Inhibition of glutathione synthesis augments lysis of murine tumor cells by sulfhydryl-reactive antineoplastics.

GSH plays an important role in cellular defense against a wide variety of toxic electrophiles via the formation of thioether conjugates. We studied the role of GSH in murine tumor cell defense against a novel class of sulfhydryl-reactive antineoplastics, the sesquiterpene lactones (SL). Incubation of P815 mastocytoma cells with any of the four SL tested (vernolepin, helenalin, elephantopin, and eriofertopin) for 1 h resulted in 70-97% depletion of GSH. The importance of GSH resynthesis upon exposure of tumor cells to SL was evaluated with the use of buthionine sulfoximine (BSO), a selective, nontoxic inhibitor of gamma-glutamylcysteine synthetase. Inhibition of GSH synthesis with 0.2 mM BSO markedly enhanced SL-mediated cytolysis of four murine tumor cell lines. A 6- to 34-fold reduction in the amount of SL causing 50% lysis was obtained with BSO. Addition of BSO to P815cells either during or immediately after a 1-h pulse with 10 micrograms/ml of vernolepin increased cytolysis from less than 3% to 78-82%. However, a 1.5-h delay in the addition of BSO to such cells, which allowed for substantial resynthesis of GSH, reduced cytolysis to 30%. Recovery of GSH synthetic capacity after BSO treatment correlated with loss of the synergistic effect of BSO on lysis by vernolepin. BSO did not augment cytolysis by six other antineoplastics (doxorubicin, mitomycin C, vinblastine, cytosine arabinoside, maytansine, and 1,3-bis-[2-chloroethyl]-1-nitrosourea [BCNU]). Of these, only BCNU depleted cellular GSH. Lysis by jatrophone, another GSH-depleting antitumor agent, was increased 21-fold by BSO. Since prolonged incubation with BSO alone results in near-complete GSH depletion without loss of cell viability, SL-mediated cytolysis is probably not a result of GSH depletion. We have demonstrated, however, a critical role for GSH synthetic capacity as a determinant of tumor cell susceptibility to cytolysis by SL. GSH also plays an important role in cellular defense against oxidative injury. Vernolepin, acting as a GSH-depleting agent, markedly sensitized tumor cells to lysis by H2O2 (greater than 6.5-fold increase with 20 micrograms/ml of vernolepin). These findings suggest the possibility that the coordinated deployment of sulfhydryl-reactive antitumor agents, BSO, and oxidative injury might constitute an effective chemotherapeutic strategy.

A recessive cellular mutation in v-fes-transformed mink cells restores contact inhibition and anchorage-dependent growth.

A contact-inhibited revertant of mink cells transformed by the Gardner-Arnstein strain of feline sarcoma virus was isolated by fluorescence-activated sorting of cells stained with the mitochondria-specific dye rhodamine 123. The revertant cell line exhibited a decrease in its proliferative rate and saturation density and a complete loss of its capacity for anchorage-independent growth, but it remained tumorigenic when inoculated into nude mice. The revertant cells retained a rescuable Gardner-Arnstein feline sarcoma provirus, expressed high levels of the v-fes oncogene product and its associated tyrosine kinase activity, manifested elevated levels of phosphotyrosine-containing cellular proteins similar to those observed in v-fes-transformed cells, and were refractory to retransformation by retroviruses containing the v-fes, v-fms, and v-ras oncogenes. Fusion of the revertant and parental cells generated somatic cell hybrids which formed colonies in semisolid medium, indicating that the block in transformation was recessive. These data together with the observation that the revertant phenotype is unstable in continuous culture suggest that the loss of transformation is due to the presence of limiting quantities of a gene product which functions downstream of the v-fes-coded kinase in the mitogenic pathway.

Isolation from a murine fibrosarcoma of cell lines with enhanced plating efficiency in vitro.

The technique for the isolation of mutants was applied to establish highly clonogenic cells from a fibrosarcoma (FSA) that had an extremely poor growth capacity in vitro (10(-6)--10(-7) of surviving fraction of cells). After consecutive clonings, the surviving fraction of cells increased to 1--5 X 10(-1), whereas that of the ordinarily maintained culture remained at a low level. Selected clones were analyzed in vitro and/or in vivo. The results indicated that the FSA was composed of heterogeneous cells or cells having a potential variability in cloning ability in vitro, metastatic ability in vivo [lung colony-forming efficiency (LCFE)], and DNA content. The relatively high DNA content of one of the clones, FSA 1233, continued after growth in vivo or in vitro, indicating its hereditary nature. FSA 1233 was also demonstrated to have a lower LCFE when the cell suspension was made from a tumor rather than from a culture in vitro. The difference of surviving fractions between the cells in the two conditions was not enough to explain the difference in LCFE's between them. These cells could grow under either in vitro or in vivo conditions and could be made readily into single-cell suspensions. The cells are, therefore, available as a system for analysis of the response of cells in vitro after in vivo treatment with various agents.

Dual-frequency proton spin relaxation measurements on tissues form normal and tumor-bearing mice.

Measurements of pulsed nuclear magnetic resonance relaxation times T1 and T2 were made at 2.7 MHz and 15 MHz on water protons in liver, kidney, spleen, muscle, and brain tissue from normal A.SW mice, and in the same tissues and tumors from A.SW mice developing MSWBS tumors (an ascites sarcoma) following dorsal sc implantation of tumor fragments. The measurement precision obtained from improved spectrometer design made it possible to show that T1 and T2 in all tissues except brain were increased by the presence of the tumor in the animal. The responses exhibited by T1 and T2 in liver and kidney were proportional to the size of the tumor. The smaller responses shown by T1 in spleen (15 MHz) and T1 and T2 in muscle (2.7 MHz) also showed a significant correlation with tumor size. The relaxation times for tumor (T1 at 2.7 MHz, T2 at 2.7 and 15 MHz) showed a significant negative correlation with tumor size: The times decreased as tumor size increased. The results were analyzed by use of the two-phase fast exchange model and were consistent with the effects expected if tissue water content increased and tumor water content decreased as tumor size increased. The analysis indicated that the effects arose primarily through changes in b, the fraction of water bound to fast exchange sites on the protein, with important modifications from changes in the correlation times Tc and Tm;Tr controlled the frequency that must be chosen for specific diagnostic applications.

Purification and characterization of a lipolytic factor (toxohormone-L) from cell-free fluid of ascites sarcoma 180.

A lipolytic substance in the ascites fluid of mice with Sarcoma 180, called toxohormone-L, was purified and characterized. The lipolytic activity of toxohormone-L was measured in vitro using rat adipose tissue slices. Toxohormone-L, purified approximately 90-fold from the ammonium sulfate fraction, gave a single band on polyacrylamide gel electrophoresis. Its molecular weight was about 75,000, and its isoelectric point was 4.7. Toxohormone-L is heat labile and nondialyzable, but, on its digestion with trypsin, an active fragment that was heat stable and dialyzable was produced. Toxohormone-L is a protein and is also present in the ascites fluid of patients with hepatoma and Grawitz's tumor.

Comparative immunohistochemical reactivity of monoclonal and polyclonal antibodies to H-ras p21 in normal and neoplastic tissues of rodents and humans.

Five mouse monoclonal antibodies (mES 2, 4, 8, 13 and 20) produced against bacterially expressed BALB ras p21 and five other monoclonal or polyclonal antibodies (Cetus rabbit or mouse pan p21, RAP-5, Triton Ha-ras, Y13-259) to H-ras p21 were used for comparative immunohistochemical detection of H-ras p21 by the avidin biotin peroxidase-complex technique in selected normal fixed tissues and tumors from rats, mice, and humans. Nine of these antibodies were immunoreactive with cell membranes and cytoplasm of harvey sarcoma virus-induced sarcoma cells, but usually only with Bouin's fixed tumors. A few normal tissues were immunoreactive with some of the antibodies except for many immunoreactive tissues found with mES 13. Although mES 13 stained sarcoma cells on the cell membrane, a prominent granular staining, which appeared to be mitochondrial or lysosomal, was seen in many normal and neoplastic rodent tissues and in normal human colon, colon polyps, and carcinomas. Interpretation of positive immunoreactivity with any antibody was sometimes difficult due to nonspecific (background) staining. The cellular pattern of specific reactivity (membrane or granular) and inhibition of immunoreactivity by absorption of the antisera with H-ras p21 was therefore important. Western blots with BALB transforming (Lys 12) p21 expressed in E. coli revealed reactivity of all antibodies except for RAP-5. In addition, immunoblots of solubilized proteins from the EJ cell line with RAP-5 indicated reactivity of this monoclonal antibody with proteins of several different molecular weights. RAP-5 also never specifically immunoreacted with cell membranes of normal or malignant cells including EJ cells. Interpretation of these findings in comparison with those in published reports of H-ras p21 localization in fixed tissue sections is discussed, including the importance of fixative, specific antibody and controls.

Purification by preparative electrophoresis and characterization of histone H2B from rat chloroleukaemia.

Highly purified histone H2B from rat chloroleukaemia has been isolated by preparative electrophoresis at pH 2.7 in polyacrylamide slab gel, using the fraction F2b of Johns (Johns E. W. (1964) Biochem, J. 92, 55-59) as starting material. This histone was characterized by amino acid analysis and end groups determination. Comparative studies with homologous calf thymus histone show similarity of the amino acid compositions and of the amino terminal groups. the carboxyl terminal sequence presents two conservative substitutions.

Comparison of non-collagenous type IV collagen components in the human glomerulus and EHS tumor.

A method for the isolation of the NC1 domain of type IV collagen has been developed using the EHS sarcoma, a basement membrane-producing mouse tumor. This NC1 domain has been compared to the NC1 of human glomerular basement membrane (hGBM) to assess its usefulness in the biochemical characterization of the Goodpasture antigen which is associated with NC1. Both NC1 isolates appeared to migrate by gel filtration as hexamers (Mr 160,000) and in SDS-polyacrylamide gel electrophoresis as dimers and monomers (Mr 54,000 and 26,000), and were shown to share biochemical identity by amino acid analysis. The hGBM NC1 showed greater complexity in the monomer region, and when compared by two-dimensional gel electrophoresis was found to contain more components in both regions than EHS NC1. Anti-GBM autoantibodies from patients with Goodpasture's syndrome reacted with the EHS NC1 by immunoblotting of two-dimensional gels. The EHS NC1 isolated by reverse phase HPLC partially inhibited the reactivity of the anti-GBM autoantibodies against hGBM NC1 by inhibition ELISA assay. Reverse phase HPLC elution of EHS and hGBM NC1 showed differences in subunit composition and interaction; complete dissociation of the EHS monomers and dimers in 0.1% trifluoroacetic acid was observed, whereas hGBM monomers and dimers eluted together. Rotary shadowing of hGBM NC1 domains revealed size heterogeneity of globular domains, compared with greater uniformity of EHS NC1 hexamers. We conclude that EHS NC1 contains an epitope(s) that is reactive with human autoantibodies to hGBM NC1. However, the immune response in Goodpasture's syndrome may involve antibodies directed against epitopes which are present in greater density and on a more complex array of peptides in the hGBM NC1 than in EHS NC1.

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.

Tumoral myosins of Ni3S2-induced rhabdomyosarcomas in rat and rabbit: comparative studies with adult and fetal myosins of skeletal muscle.

Tumoral myosins were isolated from rat and rabbit rhabdomyosarcomas and compared with normal adult and fetal skeletal myosins. The synthetic filaments, the light-chain composition and the Ca2+ ATP-ase activity were studied. In the presence of Mg2+, normal myosins precipitated as bipolar filaments (0.5 micrometer), fetal and tumoral myosins, however, precipitated as long fusiform filaments (1 to 10 micron). SDS-PAGE revealed that tumoral myosins contain the same light-chains as fetal myosin (25000 and 18000 daltons, L25-L18). The third light-chain of the normal muscle myosin (16000 daltons, L16) was absent. In addition, Urea-PAGE revealed the absence of the phosphorylated form of the L18 in fetal and tumoral myosins. Ca2+ ATPase activity measurements performed in function of the Ca2+ concentration showed similarities between fetal and adult muscle myosins. The Ca2+-ATPase activity of tumoral myosins, however, was very low and slightly activated by increasing the Ca2+ concentration (0.01 to 10 mM). The investigation has shown that fetal and tumoral myosins are identical concerning the ultrastructure of their synthetic filaments and their light-chain composition. This was not so in regard to the Ca2+ ATPase activity. This is probably the result of the expression of a new myosin- or of one of its polypeptides-, which has a different Ca2+-ATPase activity.

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.

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

Detection of Raji binding activity in hyperimmune allogeneic tumor bearing sera associated with anti-BSA activity.

Brown Norway (BN) rats were implanted twice with allogeneic (Lewis strain) Moloney sarcoma tumors (LM-2) and serum samples were assessed for Raji binding activity during primary and secondary tumor growth and rejection. Maximum Raji binding was observed 25 days after a primary tumor implant; thereafter, the binding activity decreased. Accelerated tumor rejection was observed after a second tumor implant and was associated with a 3-fold increase in serum Raji binding activity which remained elevated up to 40 days post-tumor implant. Raji binding activity in hyperimmune rats co-migrated with IgG in G-200 fractionated serum. Polyacrylamide gel electrophoresis (PAGE) revealed the presence of bovine serum albumin (BSA) on Raji cell membranes which reacted immunochemically with rabbit anti-BSA antiserum. Immunodiffusion studies revealed that sera from hyperimmune rats produced a precipitin band when reacted with Noniodet P-40 (NP-40) lysates of Raji cells and formed a line of identity with BSA.

Extremely underwound chromosomal DNA in nucleoids of mouse sarcoma cells.

The superhelical properties of chromosomal DNA from cells of a mouse sarcoma were investigated in neutral sucrose gradients containing ethidium bromide. Removal of negative supercoiling from the DNA of the sarcoma cells required a substantially higher dye concentration than was necessary in the case of DNA from cultured mouse fibroblasts. The calculated value of the mean superhelical density in malignant cells (sigma = -0.14) appears abnormally high compared with the value (sigma = -0.09) obtained for DNA of mouse fibroblasts. Chromosomal DNA from mouse sarcoma cells is therefore concluded to be highly deficient in helical turns.

In vivo testing of hypoxic radiosensitizers using the KHT murine tumour assayed by the lung-colony technique.

The KHT transplantable tumour of C3H mice has been used as a model tumour for the invivo study of hypoxic cell sensitizers. Eleven sensitizers comprising four nitrofuran five nitrobenzene and two nitroimidazole derivatives, which have been shown to be effective on hypoxic mammalian cells in vitro, have been investigated. Two of these compounds, metronidazole (2-methyl-5-nitroimidazole-1 ethanol) and tinidazole (ethyl [2-(2'-methyl-5'-nitro-1'-imidazolyl) ehtyl] sulfone), showed signs of hypoxic cell-sensitization in vivo when given systemically by intraperitoneal injections. In addition, preliminary testing of the nitrobenzene NDPP (P-NITRO-3-DIMETHYL-PROPRIOPHENONE HYDROCHLORIDE) INDICATED THAT WHEN IT WAS INJECTED DIRECTLY INTO THE TUMOUR AND IRRADIATION WAS COMPLETED WITHIN TEN MINUTES AFTER INJECTION, APPRECIABLE SENSITIZATION WAS OBTAINED. More detailed studies indicated that both metronidazole at 1,500 mg/kg and tinidazole at 750 mg/kg given intraperitoneally gave an enhancement ratio of 1-5 for a chronically hyopix cell population in this solid tumour in air-breathing mice. Measures of plasma levels of metronidazole and enhancement ratios obtained in the present in vivo system seem in relative agreement with the in vitro and in vivo results of others.

The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. II. Calibration of the staining method.

The protein content of identical samples from 5 different cell types (lymphocytes of the thymus and of the bursa of FABRICIUS of the chicken, EHRLICH ascites tumor cells, YOSHIDA ascites tumor cells, and rat liver cells) have been determined both macroscopically, by the method of LOWRY (1951), and microspectrometrically, by the tetrazonium coupling method of NOHAMMER (1978). In all the different cell types, a strong correlation is shown between the protein values determined by the 2 methods. By comparing the values thus measured, a conversion factor has been obtained such that an extinction of 0.4235, determined microspectrometrically, corresponds to a protein mass of 1 pgm. To test the accuracy of the microspectrometric method of protein determination at the lowest extreme point, the protein content of rat liver mitochondria has been measured and a mean value of 0.239 pgm protein per mitochondrion obtained. This corresponds well with the value of 0.233 pgm per mitochondrion as determined macroscopically by GEAR (1972).

Identification of transformation-specific proteins synthesized in cryptovirogenic mammalian cells.

The cryptovirogenic PR-RSH-19 (H-19) hamster cell line, derived originally from a tumour induced by the virus rescued from XC cells by transfection, was studied. The pp60v-src protein was detected by immunoprecipitation with sera from tumour-bearing rabbits (TBR sera) from lysates of H-19 cells. A closely related protein p60 was precipitated by antiserum obtained from hamsters with tumours induced by H-19 cells. The pp60v-src of H-19 cells possesses an associated kinase activity. These results are consistent with the notion that the detected pp60v-src protein is a product of the cryptic sequences in H-19 cells.

Fractionation and characterization of euchromatin isolated from mouse ascites sarcoma cells.

Euchromatin specimen prepared by the usual method formed large clumps and had various shapes under electron microscopy. A method of separation of the euchromatin specimen into chromatin fractions having relatively homogeneous form was examined and partial characterization of these fractions was carried out. The heavy euchromatin fraction was a large network of thin fibrils (about 100 A in diameter) and various thick fibers. The intermediate euchromatin fraction consisted of relatively homogeneous networks of thick knobby fibers (about 250 A in diameter). The light euchromatin fraction had metworks of thick fibers. These chromatin fractions were quantitatively prepared from sonicated nuclei of mouse ascites sarcoma cells. Twenty-one or twenty-two bands of non-histone proteins besides histones were detected in these chromatin fractions by SDS-polyacrylamide gel electrophoresis. There were significant differences in the electrophoretic patterns of non-histone proteins among these chromatin fractions.