Studies on the transformation of rat embryo cells of low passage by carcinogenic fluorenylhydroxamic acids and their acetate esters

Rat embryo cells of low passage subjected to a single treatment with certain carcinogenic fluorenylhydroxamic acids and their respective acetate esters showed signs of transformation in vitro, such as changes in phenotype, growth in soft agar and agglutination with concanavalin A. In addition, certain changes in karyotype and loss of diploidy were observed. There was no evidence, either by electron microscopy or by assay of RNA-dependent DNA polymerase, for the presence of virus. None of these cell lines produced tumors after inoculation into the isologous host. The results of these study lead us to suggest that malignant transformation is a multistep process and that certain criteria of transformation of rat embryo cells are associated with the initial stage(s) in which the cells are transformed without being tumorigenic. The ultimate test for malignant transformation of rat embryo cells remains the production of tumors in a susceptible host after inoculation of treated cells.     

The co-carcinogenic activity of 4-nitropyridine-1-oxide (4-npo) and prevention of transformation by type-specific anti-viral antibodies

Summary Fischer rat embryo cells chronically infected with Rauscher murine leukemia virus, and known to be sensitive to transformation by potent chemical carcinogens, were transformed by the weak carcinogen 4-nitropyridine-1-oxide. Transformed cells grew in semi-solid agar and produced tumors in newborn Fischer rats. Transformation was inhibited by antisera specific for the ecotropic Rauscher murine leukemia virus, but not by antisera of equal toxicity specific for xenotropic Swiss mouse AT-124 virus. This work was supported by contract NO1-CP-43240 within the Virus Cancer Program of the National Cancer Institute.     

Chromosomal rearrangements and their role in spontaneous immortalization and transformation of rat embryo cells in vitro

Peculiarities of chromosomal rearrangements were studied in cells of the spontaneously immortalized LRec-1 and LRec-3 lines derived from rat embryo fibroblasts, as well as in LRec-1k clone cells and LRec-1sf line cells with autocrine regulation of proliferation at various cell transformation stages. The lines were obtained from rat embryo fibroblasts by cloning during rapid aging of the cultures. Using the G-banding of chromosomes, it was shown that in the process of transformation, cells of the LRec-1 and LRec-3 lines as well as of LRec-1sf maintained diploidy and specific clonal rearrangements of chromosomes 7 and 19, which were revealed earlier at the immortalization stage. In the LRec-1 cells, new clonal rearrangements of chromosomes 10 and 20 were observed, while rearrangements of chromosomes 1, 2, 11, 15, 18, and 19 were observed in the LRec-1sf cells. In the LRec-3 cells, as well as in cells of the LRec-1k clone, new chromosome rearrangements were absent. Loci involved in chromosomal rearrangements were compared with the genes located in them according to RATMAP data. The role of rearrangements of chromosomes 7 and 19 in the immortalization and malignant transformation of embryo fibroblasts is discussed, as well as the roles of other chromosomes during acquisition of the specific signs of the transformed phenotype by the LRec-1 and LRec-1sf cells.     

Species specificity affects the choice of S9 preparations for use in the hamster embryo cell transformation system

Summary Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolizeN-2-acetylaminofluorene to 2-aminofluorene andN-hydroxy-acetylamino-fluorene, products that transform hamster embryo cells. Large amounts ofN-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels ofN-hydroxy-acetylaminofluorene were formed after incubation ofN-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested withN-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system. This work was supported by Contract N01-CO-75380 with the National Cancer Institute, NIH, Bethesda, MD 20205.     

Influence of short-chain fatty acids and osmolality on mucin release in the rat colon

Summary The influence of short-chain fatty acids (SCFA) and osmolality on mucin release in the rat colon was studied histochemically by determining number of stained mucin-containing cells. SCFA did not significantly influence the number of cells staining for mucin. Hypertonic solutions (360 mosm/l) did not affect mucin release in the proximal colon, but stimulated mucin release in the distal colon. Solutions of lower osmolality (300 or 250 mosm/l) caused a considerable release of mucin from goblet cells as well as vacuolated cells in both the proximal and the distal colon; the lower the osmolality, the more mucin was released. The mucosa of the distal colon was conspicuously affected by solutions of lower osmolality. The influence of osmolality on mucin release was entirely local.Supported by a grant from the Deutsche Forschungsgemeinschaft (En 65/9)The authors wish to thank Prof. H. Höller and G. Rechkemmer for critical advice and Miss G. Becker for technical assistanceA preliminary portion of this study was presented at the 3rd Meeting of the European Intestinal Transport Group, Southampton, 21.–23. April, 1980     

Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro

Summary Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests. Research sponsored by the National Cancer Institute under Contract No. N01-CO-75380 with Litton Bionetics, Inc.     

Autocrine growth factor and spontaneous transformation of rat liver epithelial cells

Summary Liver epithelial cells from a young rat were cultured and passaged for a long period until they showed a malignant transformation (tumorigenicity in athymic mice), to know when an autocrine growth factor is produced in the course of a spontaneous malignant transformation of normal cells in vitro. The cells initially showed a cellular transformation and an increased growth rate, then produced an autocrine growth factor after Passage 55, and finally the cells after Passage 105 showed a malignant transformation (tumorigenicity in vivo). It is suggested that in addition to an autocrine growth factor, some other factors might be related to a malignant transformation (tumorigenicity in vivo).     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.     

Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.     

华山新麦草胚和胚乳的发育研究

采用常规石蜡切片法,观察了华山新麦草胚和胚乳的发育过程,结果表明,华山新麦草胚和胚乳的发育过程与一般禾本科植物基本相同,胚胎发生属紫宛型,顶细胞和基都参与胚体的形成,胚胎发育经过二细胞原胚,多细胞原胚,球形原胚,梨形原胚,分化胚和成熟胚阶段,成熟胚具有胚根,胚芽,盾片,胚牙鞘,胚根鞘,外胚叶等典型禾本科植物成熟胚的结构,胚乳发育类型为核型,包括游离核阶段,细胞化阶段和生长成熟阶段,待大量游离核形成之后才形成细胞壁,紧贴胚囊的一层胚乳细胞最后形成种子的糊粉层,其余的胚乳细胞最后充满淀粉粒,其特点为：（1）有双球形原胚的现象;（2）反足细胞解体较早;（3）胚乳游离核时期和细胞时期胚乳细胞核的核仁多样。     

Transforming activities of trichloroethylene and proposed industrial alternatives

Summary Three chlorinated hydrocarbons, proposed or already in use as industrial subsitutes for the hydrocarbon trichloroethylene, were tested for in vitro transforming potential in a Fischer rat embryo cell system (F1706), which previously has been shown to be sensitive to transformation by chemical carcinogens. Trichloroethylene and the three substitutes (1,1,1 trichloroethane, tetrachloroethylene and methylene chloride) all were found to induce transformation, the three substitutes being equal or more efficient transforming agents. This work was supported by Contract N01-CP-43240 within The Virus Cancer Program of the National Cancer Institute.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.     

Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation

The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3–11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells. This work was supported by the National Natural Science Foundation of China (grant no. 30571336) and the President Foundation of the Agricultural University of Hebei.     

In vitro transformation of cheno- and ursodeoxycholic acids and their 7-oleyl esters by human intestinal microflora

Chenodeoxycholic and ursodeoxycholic acid are used widely for the treatment of gallstones. A possible drawback to their utility is their conversion to lithocholic acid, which has displayed histotoxicity and mutagenicity. The 7-oleyl esters of cheno- and ursodeoxycholic acid are not degraded by fecal bacteria and may represent safer means of treatment.     

Rimantadine but not amantadine protects fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene

Summary The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimantadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene. This work was supported by Contract N01-CP-43240 within The Virus Cancer Program of the National Cancer Institute.     

Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.     

Developmental potential and behavior of tetraploid cells in the mouse embryo

Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.