Formation of colonies of P-388 leukemic cells in semisolid agar

Substrain P-388/A2 adapted to cultivation of agar gel in the form of compact colonies was obtained as a result of alternating passages of cells of ascitic mouse leukemia P-388 in the primary semifluid agar culture and in the mouse abdominal cavity. The efficacy of colony formation and the size of the colonies depended on the initial density of the cell suspension. In case of introduction into the agar medium of 100 cells/ml the planting efficacy constituted 20%, and the number of cells in the colony by the 8th--10th days of cultivation reached 13 000.     

The expression of P-glycoprotein in leukemia P388 cells with induced doxorubicin resistance]

beta-D-galactose-containing glycoproteins were prepared from cells P-388 leukemia and from P-388 leukemia cells with induced resistance to doxorubicin. It was shown by HPLC method that plasma membranes from resistant cells contain 4-4.5% P-glycoproteins and plasma membranes from sensitive cells contain P-glycoproteins about 10 times lower.     

Cell-cycle specific cytotoxicity mediated by stizophyllin (2 alpha,3 beta,12 beta-trihydroxypregna-4,7,16-trien-20-one), a novel electrophilic pregnane isolated from Stizophyllum riparium

A structurally-novel pregnane derivative, 2 alpha,3 beta,12 beta-trihydroxypregna-4,7,16-trien-20-one (stizophyllin), was isolated from an extract of Stizophyllum riparium (H.B.K.) Sandw. on the basis of bioactivity-guided fractionation and confirmed to mediate a potent cytotoxic response with cultured P-388 cells. We presently report a detailed isolation procedure and the results of studies designed to examine its mechanism of action. By means of a Michael-type addition, stizophyllin formed adducts with nucleophilic substances such as L-cysteine and beta-mercaptoethanol. The adduct with beta-mercaptoethanol was isolated, structurally characterized, and found to be 20-fold less cytotoxic than stizophyllin. Stizophyllin interacted with DNA, but no mutagenicity was observed with Salmonella typhimurium strain TM677 or cultured Chinese hamster ovary cells, and no in vitro reaction occurred with guanosine. Relative to control cell cultures, the total biosynthesis of DNA, RNA or protein was reduced when P-388 cells were treated with stizophyllin. However, the cell number did not increase in the presence of inhibitory stizophyllin concentrations (e.g., 4 micrograms/ml), and the DNA content (per cell) actually doubled after approximately 48 h. Consistent with this, stizophyllin blocked the cells in the G2 + M compartment of the cycle, and the block appeared to be specific for the G2 phase. Accordingly, stizophyllin did not modulate in vitro tubulin polymerization reactions nor did it affect the morphology of dibutyryl cAMP-treated astrocytoma cells in culture. These data suggest that the cytotoxic activity of stizophyllin is mediated by covalent reaction with a cellular component (such as a sulfhydryl-containing protein) by means of a Michael-type addition. Based on the cell-cycle specificity of the response, it appears that this interaction prevents the cells from progressing through mitosis.     

Antitumor activity of the components of a carminomycin complex

The antiblastomic activity of the carminomycin complex components was studied with respect to 8 strains of transplantable tumors of mice: lymphosarcoma L10-1, prestomach cancer OZh-5, sarcoma 180, lymphoid leucosis L 1210, lung bronchogenic cancer RL, lymphodenosis NK/LI, Ehrlich carcinoma and Garding-Passy melanoma. It was shown that components I, II and III possessed almost the same high antiblastomic activity and the same optimal administration schemes should be used for them. The scheme consisted of two-fold administration of the drug at intervals of 96-120 hours. Component I had broader therapeutic ranges and was more active against the lung bronchogenic cancer as compared to component II. All 3 components had no selective antiblastomic effect on the ascitic form of Ehrlich carcinoma. A comparative study of the component toxicity and pharmacology is required for final conclusion as to the recommendation of one of the components for clinical trials.     

Synthesis and antitumor properties of 3'-desamino-3'-dimethylformamidinorubomycin

Interaction of rubomycin (daunorubicin) chlorhydrate with dimethylformamidine diethyl acetal yielded 3'-desamino-3'dimethylformamidinorubomycin chlorhydrate (DFR). Comparative antitumor activity of DFR and rubomycin was studied on mice with respect to ascitic lymphadenosis NK/Ly and Ehrlich carcinoma, hemocytoblastosis La, leukemia P-388 and two solid tumors i. e. lymphosarcoma LIO-I and sarcoma 180. The highest antitumor effect of DFR was observed in the mice with Ehrlich carcinoma and lymphadenosis NK/Ly after the drug intravenous administration for 4 times. By selectivity of the antitumor effect DFR was inferior to rubomycin with respect to lymphosarcoma LIO-I and sarcoma 180. It was shown that the antileukemic activity of DFR and rubomycin with respect to hemocytoblastosis La was practically the same. In the experiments with leukemia P-388 DFR was inferior to rubomycin.     

Infection of a macrophage-like cell line, P388D1 with reovirus; effects of immune ascitic fluids and monoclonal antibodies on neutralization and on enhancement of viral growth

We studied the effect of antibody on the growth of reovirus, serotypes 1 and 3, in P388D1, a continuous mouse macrophage-like cell line. Enhanced growth of virus was observed when cells were infected in the presence of nonneutralizing monoclonal antibodies or subneutralizing concentrations of either immune ascitic fluids or neutralizing monoclonal antibodies. Both enhancement of viral growth and neutralization were accompanied by an antibody-mediated increase in binding of radiolabeled virus to P388D1 cells. Although neutralization was seen only with monoclonal antibodies directed toward the sigma-1 surface protein of the virus, enhancement was observed with two monoclonal antibodies directed toward other surface proteins. Trypsin treatment of P388D1 cells abrogated enhanced growth of virus mediated by a mouse IgG2a antibody; preincubation with P388D1 with human IgG1 but not IgG2 myeloma proteins also abrogated enhancement by immune ascitic fluid or monoclonal antibody. These observations are compatible with known properties of P388D1 Fc receptors and support the role of the Fc receptor in antibody-mediated infection.     

Investigation of sesquiterpene lactones as protein synthesis inhibitors of P-388 lymphocytic leukemia cells

The mode of action of helenalin and bis(helenalinyl) malonate as protein synthesis inhibitors of P-388 lymphocytic leukemia cells was investigated. The initial characterizations were carried out in crude lysates of the P-388 cells. In the lysate, there was a 4 min lag after the addition of drug before inhibition of protein synthesis occurred. Both drugs allowed run-off of preformed polysomes, but did significantly inhibit the formation of the 80 S initiation complex suggesting a preferential inhibition of one or more initiation reactions. The effect of these drugs on inhibition of both elongation and initiation reactions was further investigated using more fractionated systems prepared from P-388 cells. Poly(U)-directed polyphenylalanine synthesis was marginally inhibited by both drugs, but the degree of inhibition was not sufficient to explain the inhibition observed in either the lysate or in whole cell preparations of P-388. The formation of the ternary initiation complex was not significantly inhibited by either drug, but the conversion of this complex to the 48 and 80 S initiation complexes was inhibited. The inhibition of 48 S initiation complex formation by both drugs was sufficient to explain their inhibition of protein synthesis in whole cells.     

Differential stabilization of cytochrome P-450 isoenzymes in primary cultures of adult rat liver parenchymal cells

Summary Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450sp and presumablyc were well preserved, P-450a was reduced but clearly measurable, P-450h was totally lost whereas P-450sb ande were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.     

Configuration of surfaces of human cancer cells in effusions. A scanning electron microscopic study of microvilli.

The surfaces of viable cells of metastatic human carcinomas of various histologic types and primary origin, suspended in pleural and ascitic fluids, were shown by scanning electron microscopy to be covered by microvilli of variable configuration and distribution. Microvilli of some cancer cells appeared biologically active since they were capable of forming extensions and anastomoses when settling on glass. The possible specificity and significance of microvilli in the light of the experimental data were discussed.     

Conversion of a tolerogenic to an immunogenic signal by P388AD.2 cells, a lymphoid dendritic cell-like tumor line

The lymphoid dendritic cell-like tumor P388AD.2 is capable of converting a tolerogenic signal into an immunogenic one. In the present study, adherent P388AD.2 cells were pulsed with the tolerogen, fluoresceinated (FL) sheep gamma-globulin (SGG), washed, and incubated with normal spleen cells. After 24 hr, the spleen cells were harvested and challenged in secondary cultures with immunogenic doses of FL-thymic independent (TI) antigens. The plaque-forming cell response on day 3 of secondary culture was increased as much as 400% compared with control spleen cultures exposed to untreated P388AD.2 cells. The increased response was specific for the FL-hapten and occurred only when P388AD.2 cells were pulsed with FL-Ig or FL-F(ab')2 fragments, but not with FL-synthetic tolerogens or other FL-antigens. Furthermore, the augmentation required histocompatible T cells in the primary cultures. Additional experiments showed that if cultures were devoid of Lyt-1+ cells but not Lyt-2+ cells, no augmentation occurred. A variety of other macrophage-like tumor cells, some with known antigen presenting properties, were tested for the ability to present tolerogen in an immunogenic fashion. Only an I-A+ J774 clone was able to present tolerogen in an immunogenic fashion. However, we failed to find a correlation between the presence of surface Ia antigen and tolerogen-presenting ability. The results suggest that certain types of cells may play a role in immune regulation by abrogating the tolerogenicity of Ig tolerogens via their presentation in an immunogenic mode.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

Cytotoxin production by a merineLyngbya strain (cyanobacterium) in a large-scale laboratory bioreactor

A cytotoxic compound was produced by the marine cyanobacteriumLyngbya sp. Pearl strain in large laboratory-scale batch cultures. Adsorption and fractionation of methanol extracts with reverse phase (C-18) cartridges provided a rapid method for removal of bioassay interference from salts, biopolymers and pigments and concentration of the cytotoxic principles. Cytotoxicity to the murine leukemia cell line P-388 was produced in two cycles coinciding with the initiation of exponential growth and again during the late exponential growth phase. Antiviral activity against influenza virus PR8 was found in extracts prepared from early exponential growth phase cells but antiviral activity was not detected in extracts of mid-log or late-log growth phase cells. These differences in bioactivity suggests that the cytotoxic principles produced during early and late exponential growth may be different compounds. Cytotoxicity assays using murine P-388 leukemia indicates that the semi-pure compound has an IC₅₀ of < 0.25 μg ml⁻¹ to this cell line. P-388 cytotoxicity in cell extracts increased during the late exponential growth phase and the specific yield was estimated at approximately 0.14 mg g⁻¹ (dry cells).     

Effect of transforming growth factor beta-1 on ovine satellite cell proliferation and fusion

We have evaluated the effect of transforming growth factor beta-1 (TGF beta-1) on proliferation and fusion of cultured ovine satellite cells isolated from 5-month-old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta-1 inhibited fusion of these cells in a dose-dependent manner with half-maximal inhibition occurring at .08 ng/ml. Maximal inhibition (95% suppression) occurred between .1 and .5 ng/ml. TGF Beta-1 (.05-3.0 ng/ml) did not inhibit proliferation of cultured ovine satellite cells in serum-containing medium or in serum-free defined medium. In contrast, TGF beta-1 did significantly suppress serum-stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta-1 than does proliferation of either porcine or bovine satellite cells.     

Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase,and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver

Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P₁-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P₁-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of ¹⁴C-heme in microsomes prepared from hepatocytes derived from rats given 5¹⁴[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.     

Primary culture of medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>) testis: a test system for the analysis of cell proliferation and differentiation

An in vitro test system using primary testis cells of the medaka (Oryzias latipes) was established that provides quantitative data on cell proliferation and spermatocyte differentiation. The primary cultures were characterised over a period of 2 days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained at a dynamic equilibrium in vitro for several days. The proliferating cells were predominantly present amongst the clusters of suspended cells as determined by BrdU labelling (cytological identification and quantification by ELISA). Based on cytological criteria the proliferating cells were mostly spermatogonia and preleptotene spermatocytes. Differentiation of spermatocytes to spermatids or spermatozoa was also observed mainly amongst suspended cells. Quantification of cell proliferation and cell differentiation by flow cytometry was achieved by labelling the primary cells with carboxyfluorescein diacetate N-succinimidyl ester, which allowed the identification and quantification of meiotically or mitotically dividing primary cells. Addition of the flavonoid genistein (10 µg/ml) to the primary cultures inhibited both cell proliferation and cell differentiation significantly. The test system is suitable for the study of the effect of substances which interfere with spermatogenesis in the vertebrate medaka model.     

Diacridines: bifunctional intercalators. II. The biological effects of putrescine, sperimidine and spermine diacridines on HeLa cells and on the L-1210 and P-388 leukemia cells.

The effect of putrescine, spermidine and spermine diacridines on the growth of HeLa cells and of P-388 and L-1210 leukemia cells has been evaluated and compared to that of the parent compound, 9-aminoacridine. The diacridines are more effective growth inhibitors than 9-aminoacridine. The primary site of action appears to be the inhibition of RNA synthesis.     

Synthesis and antitumor activity of s-tetrazine derivatives

Fifty-five compounds of s-tetrazine derivative including hexahydro-, 1,6-dihydro, 1,4-dihydro-, 1,2-dihydro- and aromatic s-tetrazine were prepared. Their antitumor activities were evaluated in vitro by MTT method for P-388 cell and SRB method for A-549 cell. The results show that there are 9 compounds which in 10(-6) microM have more than 50% inhibition rate to A-549 cancer cell growth, and 7 compounds in 10(-6) microM have more than 50% inhibition rate to P-388 cancer cell growth. The IC(50) of compound 3q for P-388, Bel-7402, MCF-7 and A-549 are 0.6 microM, 0.6 microM, 0.5 microM and 0.7 microM, respectively. So s-tetrazine derivative is a kind of compound which possesses potential antitumor activities and is worth to research further.     

Synthesis and biological evaluation of new spin-labeled derivatives of podophyllotoxin

In order to find compounds with superior bioactivity and less toxicity, a series of spin-labeled podophyllotoxin derivatives were synthesized and tested for the partition coefficients and cytotoxicity against P-388 and A-549. Furthermore, we also determined antioxidant activities of target molecular in tissues of SD rats by the TBA method. Results revealed that most synthesized compounds showed more significant cytotoxicity against P-388 and A-549 in vitro than VP-16. Among them, 9d exhibited most potent cytotoxicity against P-388 and A-549 cells (IC50 is <0.01 and 0.13 microM, respectively). Also, the antioxidative activities showed that the modified compounds of 4'-demethylepipodophyllotoxin (9a-d and 10a-c) are higher than those of podophyllotoxin series (8a-d). The relationship between the cytotoxicity and antioxidative activity discussed.     

Cytogenetic study of spontaneous lympholeukemia in AKR mice in the process of its transplantation to an isogeneic line of animals]

Spontaneous leucosis was cytogenetically studied in subsequent generations (from 1 to 150) of AKR mice. By means of the differential staining technique of chromosomes, large variations in chromosome numbers were found in the karyotypes of leucotic cells of different generations, and the formation of cell clones containing different marker chromosomes as well as the dominance of a hyperdiploid clone with 41-42 chromosomes was revealed. Chromosome analysis of such hyperdiploid cells of the 150th generation has indicated that the supernumerary chromosomes (in 88.0% of cases examined) belong to the smallest chromosomes of the mouse karyogramm (to the 18-19th chromosome pairs or to chromosomes smaller than those of 19th pair). Similar trisomy was also observed in hypodiploid and pseudodiploid leucosis cells. It is suggested that the cell clone with trisomy for the smallest chromosomes is specific to the spontaneous lympholeucosis in AKR mice as well as to the leucosis transplanted to isogenic mice for a number of subsequent generations. Increased rate of hyperdiploid cells was associated with a generalization of leucosis. It was concluded that the development rate and the severity of transplanted lympholeucosis in AKR mice was determined by the domination of the cell clone with trisomy for the 18-19th chromosome pairs in the population of leucotic cells.