Mouse Plasmocytoma Research Articles

Essential oils of the Oleoresins from Protium Heptaphyllum Growing in the Brazilian Southeastern and their Cytotoxicity to Neoplasic Cells Lines

Two different samples of the essential oil resin harvested from two trees of Protium heptaphyllum, growing in the Brazilian southeast seashores, were obtained by hydrodistillation and analyzed by GC and GC-MS. Monoterpenes were highly predominant in both compositions of the oils. The fresher sample, harvested from the tree A, showed the predominance of myrcene (35 %), α-pinene (27 %) followed by sabinene (11 %) and β-caryophyllene (7.2 %). Terpinolene (28 %) and p-cymene (16 %) were the largest components in the trunk resin tapped by mechanical hurt-induction from the tree B. Both samples were assayed in vitro for their ability to inhibit the growth of four different of neoplasic cell. The best results were obtained from the terpinolene/p-cymene chemotype, where inhibition of 67 %, 63 % and 59 % of mouse plasmocytoma (SP2/0), neuroblastome (Neuro-2A) and monocytic cell (J774) lineages were achieved, respectively. The growth of normal cell lineage MK2 (monkey kidney epitelial cells) increased about 50 % under treatment with the oils. The effects observed on neoplasic lineages were correlated to the presence of these monoterpenes.

Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium

The resins and leaves of species of Protium are commonly used by folk medicine. In the present study, we analyse the pharmacological effects of essential oils obtained by steam distillation (leaves and resin) from Protium species. Analysis by gas chromatography (GC) coupled to mass spectrometry and retention indices calculations demonstrate that the resin oil is constituted mainly of monoterpenes and phenylpropanoids: α-terpinolene (22%), p-cymene (11%), p-cimen-8-ol (11%), limonene (5%) and dillapiol (16%), whereas sesquiterpenes predominate as the volatile constituents of the leaves. The resin of Protium heptaphyllum (PHP) and leaves of P. strumosum (PS), P. grandifolium (PG), P. lewellyni (PL) and P. hebetatum (PHT) were screened for anti-inflammatory activity by the use of mouse pleurisy model induced by zymosan (500 μg/cavity) and lipopolysaccharide (LPS) (250 ng/cavity), for antinociceptive effect (by means of preventing mice abdominal writhings), as well as NO production from stimulated macrophages and proliferation of neoplasic cell lines: Neuro-2a (mouse neuroblastoma), SP2/0 (mouse plasmocytoma) and J774 (mouse monocytic cell line). The oils from PHP, PS and PL were able to inhibit protein extravasation but no sample inhibited total or differential leucocyte counts after administrating p.o. (100 mg/kg) 1 h before stimulation with zymosan. The oils from PG, PL and PHT inhibited neutrophil accumulation whereas PHP and specially PL inhibited LPS-induced eosinophil accumulation in mouse pleural cavity. PHT was also able to inhibit mononuclear cells accumulation. Antinociceptive effect was not observed, when animals received oral administration of the essential oils (100 mg/kg). In vitro treatment with essential oils (100 μg/well) changed the NO production from stimulated mouse macrophages. PHP inhibited in 74% and PS in 46% the LPS-induced NO production. In contrast, treatment with PL was able to increase in 49% the NO production. Cell lines proliferation was affected by the oils assayed in the range of 60–100% for Neuro-2a, 65–95% for SP2/0 and 70–90% for J774. Taken together these results showed that essential oils could be useful as efficient pharmacological tools.

Effects of Repeated Exposure to High-Voltage Electric Discharges and Low-Frequency Electromagnetic Fields on Cultured Mouse P3×63Ag8 Plasmocytoma Cells

When cells of the mouse plasmocytoma P3x63Ag8 were repeatedly exposed to high-voltage electric discharges (1.34 KV/cm, 1 sec between pulses; time constant, 25 μsec), they fused into multinuclear cells. Using the same procedure, myeloma cells were induced to form heterokarions by fusion with human or mouse lymphocytes, and to internalize formaldehyde-treated erythrocytes. Under optimal conditions, cells were fused extensively without substantial loss. Exposure of the cells to low-frequency (50 Hz) sinusoidal electromagnetic fields (magnetic field amplitude of 200 μT) did not appear to perturb membrane substructures in such a way as to influence the fusogenic effect induced in high electric fields.

Human melanoma targeting with alpha-MSH-melphalan conjugate.

A conjugate made of alpha-MSH as a drug carrier and melphalan has been designed in order to target human melanoma cells. Iodination of the alpha-MSH moiety led to a relatively stable tracer which could be easily separated and analysed by reverse phase high pressure liquid chromatography. The conjugate was found to be unstable at neutral pH and a serious denaturation can take place at concentrations exceeding 100 micrograms/ml, especially in plasma. Receptor-mediated cytotoxicity has been shown by the use of cultured alpha-MSH receptor positive/negative cells as well as in vivo B16 murine melanoma model. Body distribution and uptake of the labelled compound were unaltered as compared to those of labelled free hormone. alpha-MSH receptor recognition properties also remained unchanged with a better apparent affinity of the conjugate probably due to the alkylating activity of melphalan itself. Using human melanoma dendritic cells expressing more than 10,000 alpha-MSH binding sites per cell as an in vitro model, we were able to demonstrate higher cytotoxicities as compared to melphalan-treated cells. In contrast, melanoma cells with low receptivity did not show higher cytotoxicity. P388D1 mouse plasmocytoma cells lacking receptors were much more sensitive to melphalan than the conjugate. This phenomenon appeared to be related with the number of binding sites expressed at the time of the experiment as well as cell differentiation and the doubling time. Our findings strongly support the concept of a receptor-mediated cytotoxicity and may enable the in vivo melphalan delivery to target tissues to be increased, achieving an improvement of drug penetration inside melanoma cells.

Studies on the cell phenotype characteristics of hybrid cells crossed between rat nucleated erythroblasts and mouse plasmocytoma (SP2/O) cell line.

The present study is designed to investigate the regulatory effect of mammalian erythroblasts, prior to naturally-occurring denucleation, on malignancy of mouse plasmocytoma cells and the possibility of reactivation of the pyknotic late erythroblast nuclei in hybrid cells crossed between rat intermediate or late erythroblasts of 15-day Wistar rat embryonic livers, and mouse plasmocytoma (SP2/O) cell lines. Results indicated that: (i) Suppression of tumorigenicity and reversion of the malignant phenotype were observed in hybrid cells in a similar way as those of cybrid cells crossed between reticulocytes and myeloma cells as we reported previously, thus providing further evidence to support the hypothesis that some regulatory substances already existed in mammalian intermediate and late erythroblasts long before nuclear extrusion. (ii) Appearance of positive histochemical reaction for hemoglobins in cytoplasm and electrophoretic bands of rat and mouse globin chains in hybrid cell lysate were identified. The transcripts of mouse globin genes could be readily detected by nucleic acid hybridization technique with mouse beta-globin gene probes. (iii) Reassuming of rat chromosome and globin gene products synthesis in hybrid cell indicates that the originally pyknotic nuclei of late erythroblasts could be reactivated to assume functional activity after cell hybridization. The mechanism of regulatory effect and its possible relation to naturally occurring denucleation in developing mammalian red blood cells were discussed.

Evidence of a role for NK cells in oxazaphosphorine-mediated tumor regression.

The present studies showed that nude mice xenotransplanted with L5222 leukemia responded as did syngeneic BD IX rats to low doses of mafosfamide or cyclophosphamide. Unlike rats, nude mice rarely showed resistance to a second tumor challenge. The observation that concurrent treatment of rats with cyclosporin A did not alter the rate of survival clearly indicated a T-cell-independent mechanism of tumor defense. The incidence of lung colonies from i.v. injected Lewis lung-tumor cells could be enhanced by a high dose pretreatment with mafosfamide or cyclophosphamide, whereas pretreatment at low doses was inhibitory. Since identical experiments carried out in NK-cell-deficient C57Bl/6 "beige" mice did not show such an effect, NK cells appeared to represent a possible effector cell in oxazaphosphorine-mediated antitumor effects. This assumption was further supported by the fact that enhanced NK cell activity could be observed in the 51Cr release assay using spleen cells from mafosfamide-treated L5222-bearing rats. The transplantation of the unrelated syngeneic ovarian carcinoma OV-342 to animals that had previously been cured of L5222 leukemia did not lead to the rejection of this tumor. This indicates that a specific resistance against L5222 leukemia had developed. In contrast, a T-cell-dependent antitumor effect was demonstrated for mafosfamide in the MOPC-315 mouse plasmocytoma. Therefore, we conclude that the effector cell for tumor rejection depends on the type of tumor. This, of course, does not exclude a common target cell for the immunopharmacological activity of oxazaphosphorines.

Initiated complexes of RNA polymerase II are concentrated in the nuclear skeleton associated DNA

Several preparations of nuclear matrices containing varying amounts of DNA were obtained from mouse plasmocytoma P3-X63-Ag8.653 cells and tested for the presence of RNA polymerase II activity. It has been demonstrated that about 25% of RNA polymerase II activity detected in the original nuclei can be recovered in isolated nuclear matrices. Only DNA-bound RNA polymerase II was found in the isolated matrices, while both free and DNA-bound RNA polymerase II activities were detected in the original nuclei. RNA polymerase II activity found in the isolated matrices did not depend on the portion of DNA recovered in the nuclear matrices in a large interval between 91 and 1.5% of DNA content in the original nuclei. The conclusion has been drawn that initiated RNA polymerase II molecules are non-randomly distributed along DNA loops. They are concentrated near the points of DNA attachment to the nuclear skeleton.

A study of some molecular and kinetic properties of two tRNA methyltransferases from mouse plasmocytoma.

A tRNA(adenine-1)methyltransferase and a tRNA(cytosine-5)methyltransferase have been partially purified from mouse plasmocytoma MOPC 173. Their apparent Mr are 200000-230000 and 110000-140000, respectively, as determined by gel filtration and density gradient centrifugation. Both enzymes exhibit maximum activity in the presence of high concentrations of monovalent cations (0.175 M and 0.25 M KCl, respectively) and in the absence of magnesium. Their kinetic constants have been determined at various KCl concentrations, with several tRNA species as substrates. These constants may differ by more than one order of magnitude, depending upon the substrate used, and they are strongly dependent upon the ionic concentration as well. The possibility that the tRNA(adenine-1)methyltransferase from mouse plasmocytoma is different from the homologous enzyme purified from a normal rat tissue [Glick, J. M. and Leboy, P. S. (1977) J. Biol. Chem. 252, 4790-4795] is discussed.

In vitro effects of cyclosporin A (CSA) on human hemopoietic cell lines

The sensitivity of 18 permanent hemopoietic cell lines to Cyclosporin A (CsA) was tested in a 3H-thymidine incorporation rate assay. Two human T cell lines (Molt4 and CEM) were significantly inhibited by a CsA concentration of 0.5 μg/ml. Not affected at all or only inhibited by 10 to 20 times higher CsA concentrations were: three human B cell lines (4413a, Daudi, Raji), a monkey B cell line (B95-8), a mouse plasmocytoma line (X63-Ag8/653), a human non-B T cell line (Reh), four human myeloid lines (HL-60, ML-1, ML-2, ML-3), a human myelomonocytic line (Karpas 230), four human monoblastic lines (U 937, SU-DHL-1, THP-1, Karpas 241) and a human erythroid line (K 562). It therefore seems that among permanently growing hemopoietic cells a cell type specificity for T cells also exists.

In vitro transcription by mouse plasmocytoma RNA polymerase II supplemented with nuclear protein fractions

Mouse plasmocytoma (MOPC 31C) nuclear proteins were mildly extracted and fractionated into four fractions. Among them, a nonhistone (CP-I) or a saline-wash (NS-I) fraction enhanced the activity of RNA polymerase II (enzyme II) purified from MOPC 31C when each of these fractions was preincubated with enzyme II and native DNA before incubation for RNA synthesis in the presence of rifamycin AF/013. The enhancing activity of these fractions depended on the preincubation time, and was heat and trypsin sensitive. These results suggest the presence of chromatin-bound and unbound protein factors capable of accelerating some preinitiation reaction of transcription catalyzed by RNA polymerase II. The kinetics profile of the rifamycin AF/013-resistant transcription enhanced by fraction NS-I was evidently different from those observed when DNase I (or II)-treated or sonicated DNA was used as template. This fact as well as inefficiency of any kind of DNA other than native eucaryotic DNA for the enhancement by fraction NS-I indicate that the enhanced transcription takes place at certain specific sites of DNA. Evidence for a direct interaction between enzyme II and a factor involved in fraction NS-I was obtained by the following three kinds of analysis: kinetics employing preincubation of enzyme II and fraction NS-I without DNA; sucrose gradient centrifugation and gel permeation chromatography; and electrophoresis under nondenaturing conditions. The results of these analyses suggest that the enhancement by this factor correlates with some structural alteration of enzyme II accompanied by a change in its surface charge.

Phosphorylated sites of chicken erythrocyte histone H5 by a cyclic AMP-independent protein kinase from mouse plasmocytoma cells

Phosphorylated sites of chicken erythrocyte histone H5 by a cyclic AMP-independent protein kinase from mouse plasmocytoma cells

Somatic cell hybrids producing antibodies specific to human fibronectin.

We have produced somatic cell hybrids between mouse plasocytoma cells deficient in hypoxanthine phosphoribosyltransferase (P3 x 63 Ag8) and spleen cells derived from mice immunized with purified human plasma fibronectin. We report herein the properties of monoclonal anti-fibronectin antibodies released by eight different clones.

Resolution and general properties of different types of ribosomal protein kinases in mouse plasmocytoma

Three different types of protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) were isolated and partially purified from a mouse plasmacytoma microsomal KCl wash fraction, then chromatographed on DEAE cellulose and phosphocellulose. The three protein kinase activities designated by protein kinase I, II and III were characterized with respect to their capacity to utilize [γ- 32P]ATP and [γ- 32P] GTP, to interact with cyclic AMP, stimulation by cyclic AMP, substrate specificity and sedimentation behaviour on glycerol gradient centrifugation. Protein kinase I was found to be cyclic AMP dependent and preferentially phosphorylated histones. Protein kinase II and III were insensitive to cyclic AMP, protein kinase II preferentially phosphorylated histones and the protein(s) of a ribosomal KCl wash fraction eluted from DEAE cellulose between 0.2 and 0.35 M KCl and termed “PPx”. Protein kinase III phosphorylated casein and ribosomal proteins to a great extent. Studies with glycerol density gradient centrifugation indicated that protein kinase I sediments as a component of about 4.4 S, protein kinase II of 4.3 S and protein kinase III of 3 S. Chromatography on phosphocellulose of the protein kinases isolated from purified free polysomes showed the same type of protein kinases as those from microsomes. So it appears unlikely that protein kinase I and II were contaminants from the cytosol.

The modification of (Na + + K +) ATPase sensitivity to ouabain after extraction of membrane constituents distinct from the enzyme

The (Na+ + K+)ATPase from mouse plasmocytoma MOPC 173 ascitic cells was found to be resistant to ouabain in purified plasma membranes as 50 % of the enzyme activity (E 1/2) was inhibited by 120 μM ouabain. After 2 treatments by a sucrose-EDTA-imidazole buffer, the plasma membrane-bound enzyme recovered in the pellet was found to be much more sensitive to ouabain inhibition (E 1/2 = 0,4 μM). The original (Na+ + K+) ATPase sensitivity to ouabain can be restored by addition of concentrated supernatants from EDTA-treated membranes plus Ca++ and Mg++ to the pellet.

Therapeutic effect of intratumoral injection of BCG and other substances in rats and mice

The experimental treatment of a rat sarcoma (McFiFi 2) by intratumoral injection of BCG2 is described. Tumors which have a mean diameter of less than 10 mm at the beginning of treatment are fully susceptible to BCG, although spontaneous regression is not observed at this stage. The effective dose of living BCG ranges from two injections of 0.1 mg to two injections of 1 mg, given IT at an interval of 7 days. The permanent cure of a proporation of the tumors may also be induced by IT injection of a similar dose of heat-killed BCG or of MER, or of 10(9) heat-killed C. parvum, according to the same schedule. Preimmunization of the rats with living BCG does not improve the efficiency of heat-killed BCG. Direct contact between the therapeutic material and the tumor cells is critical. If rats are grafted with two pieces of the same tumor in widely separated sites, the intratumoral treatment of only one of these tumors with living BCG is sufficient to induce regression of both tumors in 50% of the animals. The effect of BCG is counteracted by injection of silica or by ingestion of polaramine. The same intratumoral treatment with living BCG was applied to different rat and mouse tumors. Only McFiFi 2 tumors were cured by intralesional BCG. C3H mouse plasmocytoma 5563 was not cured by intratumoral BCG but its growth could be prevented by mixing BCG and tumor cells at the time of grafting; this tumor was considered to be of medium susceptibility. However, until there is definite proof that the two mechanisms are identical, one should consider the regression and cure of a growing tumor, and the prevention of tumor growth, as two different phenomena. The clinical treatment of human tumors resembles the first experimental procedure more closely than the second.

Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes.

A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross-contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.

Topology of repeated sequences: relationship of nuclear RNA to the repeated sequences of the main and satellite DNA in mouse plasmocytoma cells.

The topology of repeated sequences in mouse plasmocytoma DNA was studied by high-resolution CsCl density gradient centrifugation and heterogeneous nuclear RNA.DNA hybridization. Satellite region DNA of plasmocytoma cells contains additional components and hybridizes specifically with entire heterogeneous nuclear RNA molecules. A linkage is demonstrated between the A+T-rich satellite sequences and those hybridizing with heterogeneous nuclear RNA. Heavy DNA also hybridizes specifically with heterogeneous nuclear RNA molecules that show sequence similarity to heterogeneous nuclear RNA hybridized to satellite DNA. These results could suggest that part of satellite DNA became heavier after integration of some other DNA species, which could belong to a virus or to immunoglobulin repetitive genes. Dispersed, highly repetitive, short nucleotide sequences could constitute recognition sites for such a process.

Studies on polysome-membrane interactions in mouse myeloma cells.

The nature of the interaction of membrane‐bound polysomes with the endoplasmic reticulum has been studied in MOPC 21 mouse plasmocytoma tissue‐culture cells which secrete an immunoglobulin G. Treatment of microsomes at high salt concentrations (500 mM KCl) results in the detachment of 40–45% of the bound ribosomes. This released (“loose”) fraction is predominantly monosomal in contrast to the remaining “tight” polysomes. It is shown that both ribosomal fractions contain immunoglobulin light‐chain mRNA which argues against a functional distinction between them in the cell.Polysomes have been dissociated to apparently undegraded ribosomal subunits at 0°C by incubation with puromycin in the presence of a magnesium ion buffer. Application of this procedure to membrane‐bound polysomes results in partial release of ribosomal subunits from the membrane. The ribosomal material which is not so released sediments predominantly at 60 S and this fraction can be detached by subsequent washing at 500 mM KCl. Thus, by use of the polysome‐dissociating procedure together with high salt concentrations, essentially total release of the ribosomal components can be obtained.The results indicate that the salt‐labile interactions are mediated via the large ribosomal subunits and are the only anchoring forces to the membrane at early stages of protein synthesis. The tight membrane‐bound polysomes result from the additional anchoring effect of the polypeptide chains as they grow through the membrane.

Some properties of "light" cytoplasmic ribonucleoprotein of mouse plasmacytoma.

[5‐3H]Uridine incorporation into ribosomes and other postribosomal ribonucleoprotein particles has been determined in mouse plasmocytoma MOPC406 cytoplasm after cell wall disruption. It has been shown that a large part of the radioactivity after 40 to 90‐min incubation is localized in the postribosomal zone of the gradient. The question arises whether these particles are the result of nuclear leakage during cell disintegration or whether they are native to the cytoplasm. To clarify this we have compared the following characteristics of cytoplasmic and nuclear ribonucleoprotein particles: (a) sensitivity to sodium deoxycholate; (b) sedimentation patterns in sucrose gradients; (c) buoyant density values in CsCl gradient; (d) dynamics of precursors incorporation into cytoplasmic ribonucleoprotein particles RNA.All these results have been considered as supporting the cytoplasmic origin of cytoplasmic postribosomal ribonucleoprotein particles.

DNA, rRNA and RNA partition isotherms rapidly labeled in a saline solvent system

AbstractPartition isotherms of DNA, rRNA, and rapidly labeled RNA in a salt solvent system are determined. In the salt solvent system PMB (potassium phosphate buffer 1.50 M pH 7.0:2‐methoxyethanol:2‐butoxyethanol; 3:1:variable volume), equilibrated at the temperature T, the partition isotherms of animal ribosomal RNA and DNA form a series of straight lines according to the basic relation log k = B − AT(BuO), where k is the partition coefficient, B and AT are parameters depending upon the base composition, the molecular weight, the helix content, and conformation of nucleic acids, and (BuO) refers to the 2‐butoxyethanol amount in volume percent. This relation is also valid for other nucleic acid compounds (bases, nucleosides, nucleotides, oligoribonucleotides, and transfer RNA). For RNA fractions distributed in different Kirby salt solvent systems, log k is proportional to the relative levels of adenine and guanine expressed as the A/(A+G) ratio.Partition isotherms of high molecular weight RNA from mouse plasmocytoma, labeled during a few hours, are not straight lines. This behavior indicates a heterogeneous RNA population. By plotting the specific activity of this mixture (rRNA and rapidly labeled RNA) determined in the top phase of our salt solvent system as a function of the 2‐but‐oxyethanol content, we obtained a sigmoidal curve, which characterizes the nature of the RNA population investigated. Formulas are given for the calculation of the specific activities of rRNA and rapidly labeled RNA and for the calculation of their relative ratio.