Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment

Summary A shooty tumor induced by a shooter mutant of an octopine strain of Agrobacterium tumefaciens was cloned. One clone obtained (TS038) behaved aberrantly in that it grew as a shooty tumor tissue on phytohormone free medium, but did not contain octopine synthase activity. In line TS038 the genes for octopine synthase and for the enzymes involved in agropine and mannopine synthesis were present, but were not transcribed. However, the above genes became active in TS038 tumor shoots after grafting as well as after treatment with the hypomethylating agent 5-azacytidine. After an unusually long incubation period in the growth cabinet shoot cultures appeared to have developed small shoots from the top of the leaves. This unusual form of differentiation was found to be accompanied by the induction of octopine synthase activity.     

The fine structure of the neural lobe of the mouse after transplantation under the kidney capsule

Summary The neurohypophysis of donor mice was implanted under the renal capsule of the recipients. The pituicytes survived while the neurosecretory axons disappeared. The ultrastructure of the glial cells was observed seven and nine weeks after transplantation. There were no signs of phagocytotic activity although remnants of axons were still present at seven weeks. The numerous processes of the pituicytes form a network with intercellular spaces wide in younger and narrower in older implants. The cells are connected by desmosomes and gap junctions. Pituicytes as well as blood vessels preserve their organotypic appearance. The transplant thus represents an experimental model for investigations on pituicytes in vivo in the absence of neurosecretory axons.Fellow of the Alexander von Humboldt-Stiftung. On leave from Department of Histology and Embryology, Institute of Biostructure, Academy of Medicine, Pozna, Poland     

The cell cycle in the small intestine transplanted beneath the kidney capsule in syngenic mice

Summary Transplantation of a small fragment of the ileum beneath the kidney capsule in syngenic mice results in the formation of a cyst lined with proliferating intestinal epithelium. The duration of the cell cycle in this epithelium was determined (using tritiated thymidine and the FLM method) as 14.5 h, as compared with 11.5 h in the intestinal epithelium in situ. We conclude that the intestinal content has little effect on the cell cycle of epithelial cells of the small intestine.     

Glycosaminoglycan synthesis by mammary tumor spheroids

Multicellular aggregates of tumorigenic mouse mammary epithelial cells contain a hyaluronate-rich matrix, both at the aggregate periphery and within the growing spheroid. It is proposed that the establishment of a hyaluronaterich matrix is essential to spheroid growth in vitro, and that the spheroid is a good model system for analysis of this aspect of early tumor development.     

A rapid method for the large scale purification of the intermediate filament protein vimentin by single-stranded DNA-cellulose affinity chromatography

A new method is described for the purification of the intermediate filament protein vimentin from Ehrlich ascites tumor cells using single-stranded DNA-cellulose affinity chromatography. The procedure is rapid and allows the large scale isolation of the protein. Partial characterization of vimentin shows that it has a molecular weight of 58000 and an apparent pI of 5.3. It can be degraded by the vimentin-specific, Ca²⁺-activated proteinase which results in the production of a characteristic set of degradation products. The vimentin also cross-reacts with the intermediate filament protein monoclonal antibody, α-IFA.     

Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

8—MOP对体外培养人癌细胞的光敏灭活作用

本文根据MTT只能被活的增殖细胞中线粒体切断形成紫色甲(?)的原理,测定了8—甲氧基补骨脂素(8—MOP)对体外培养人癌细胞系HCT、KB和BEL细胞的光敏灭活作用。结果表明,8—MOP和UVA光照对这几种人癌细胞有肯定的灭活作用,该作用与8—MOP剂量和光照时间以及细胞种类有关;MTT法可以作为光敏剂活性检测的一种快捷方法。     

转移及非转移肿瘤移植后615小鼠血液流变学变化的研究

血道高转移瘤株FC、淋巴合并血道高转移瘤株U14、淋巴道高转移瘤株H22、非转移瘤株P615分别接种于336只纯系近交615小鼠.不同时间取血并处死动物,进行组织学及血液流变学检查.将转移瘤发展过程分为潜伏期、侵袭期、转移早、中、晚期,非转移瘤发展过程分为潜优期、增殖期、囊腔形成期及中心坏死期.本实验结果显示,不同转移能力及途径肿瘤发展的不同时期血液流变学变化规律不同,因而表明肿瘤侵袭、转移与血液流变学变化之间存在互为因果的紧密关系.其临床诊断及治疗意义被讨论.     

Possible role of cell cycle-dependent morphology,geometry, and mechanical properties in tumor cell metastasis

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37°C, an increase in cell volume from 1400 μm³ to 5700 μm³ was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the “older” cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.     

Lipopolysaccharide induction of outward potassium current expression in human monocyte-derived macrophages: Lack of correlation with secretion

Summary Although an outwardly rectifying K⁺ conductance (I _K, A) is prominently expressed in human alveolar macrophages, the expression of this conductance in human monocyte-derived macrophages (HMDMs) is rare. We have analyzed the induction of the expression of I _K, A in voltage-clamped, in vitro differentiated HMDMs by a number of stimuli which produce either priming or activation of macrophages. Cultures were stimulated with lipopolysaccharide (LPS, 2 g/ml), interleukin 2 (IL-2, 100 U/ml), or combinations of LPS and either recombinant interferon-gamma (-IFN, 10 U/ml), phorbol myristate acetate (PMA, 0.01 or 1 g/ ml) and platelet activating factor (PAF, 20 ng/ml) for periods of up to 24 hr. Treatment of the cells with either LPS or IL-2 greatly enhanced the frequency of current expression. Treatment with either PMA or -IFN alone did not induce current expression; treatment of the cells with a combination of LPS and either PMA, -IFN, or PAF did not enhance current expression over that observed with LPS alone. The expression of the outwardly rectifying K⁺ current was observed in 36% (n=321) of the cells for cultures treated with LPS and 33% (n=55) of the cells for cultures treated with IL-2. The inactivating outward K⁺ current was absent in cells which were not treated with either LPS or IL-2. The kinetics of current activation and inactivation appeared identical to that previously described for the transient-inactivating outward current of the human alveolar macrophage. Cycloheximide (1 g/ml), an inhibitor of protein synthesis, completely suppressed LPS-induced current expression. No correlation was found between peak current amplitude and cell size in LPS-activated cells expressing the outwardly rectifying K⁺ current, indicating that current density was not held constant from cell to cell. The coupling of ion channel expression and secretion in individual HMDMs was studied using the reverse hemolytic plaque assay. Although an enhancement of K ⁺ current expression was observed following either LPS or IL-2 treatment, a quantitatively similar and uniform increase in the percentage of either IL-1 or lysozyme-secreting cells was not observed. The frequency of current expression in cells identified as secreting tumor necrosis factor- (TNF-), interleukin 1 (IL-1), or lysozyme was the same or decreased over that observed for nonsecreting cells. Thus, LPS treatment increases the number of K⁺ channels on HMDM membranes; however, K⁺ channel expression alone was not sufficient to give rise to enhanced secretion in LPS-activated macrophages. Enhanced K⁺ channel expression appears to be a part of the primary activation signal. K⁺-channel activation would hyperpolarize the membrane potential, potentially providing the driving force for calcium entry through voltage-independent pathways activated by the subsequent binding of soluble substances to membrane surface receptors, the secondary signal linked to secretion.This work was supported by NIH grant RO 1 GM36823.