Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.     

High-Yield Method for Dispersing Simian Kidneys for Cell Cultures

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique.     

Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses

Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.     

Molecular basis of toxicity of Clostridium perfringens epsilon toxin

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.     

Use of thermitase in the cultivation of embryonal mouse fibroblasts

Thermitase has been investigated as a means for obtaining single cells from tissue material, for enzymatic detachment of cultivated cells from the substrate and rarification of cells with subsequent passaging of mouse embryonic fibroblasts. The action of this enzyme was compared with that of "trypsin for cell cultivation". Tissue digestion showed that thermitase at 1/50 of the enzyme concentration needed with trypsin, was 1.5 fold more effective in yielding single cells. In cell cultivation thermitase is able to detach cells from the substrate at the same low concentration of 0.05 mg enzyme/ml and to give sufficient rarification, without the need of adding complexing agents. Rate of attachment, cell form and multiplication in subcultures corresponded to those after application of trypsin. The best results in cell detachment and rarification and the most uniform cell morphology were obtained with thermitase at a concentration of 0.025 mg enzyme/ml under addition of 4 mM of complexing agent. At that, thermitase proved 50 fold more effective than trypsin. Another advantage of thermitase is its better storage quality at 4 degrees C in dissolved form.     

Production of soluble matriptase by human cancer cell lines and cell surface activation of its zymogen by trypsin

The membrane-bound serine proteinase matriptase, which is often released from the plasma membrane of epithelial and carcinoma cells, has been implicated to play important roles in both physiological and pathological conditions. However, the regulatory mechanism of its activity is poorly understood. In the present study, we examined expression and activation state of soluble matriptase in 24 human cancer cell lines. Soluble matriptase was detected in the conditioned media from all of 5 colon and 4 breast carcinoma cell lines and 8 of 10 stomach carcinoma cell lines tested. Only two of five lung cancer cell lines released the matriptase protein into the culture media. Out of the five matriptase-negative cell lines, two cell lines expressed the matriptase mRNA. Among 24 cancer cell lines tested, 13 cell lines secreted trypsin in an active or latent form and all of them released matriptase. Most of the 24 cell lines released a latent, single-chain matriptase of 75 kDa as a major form, as well as low levels of complex forms of an activated two-chain enzyme with its specific inhibitor HAI-1. Thus, these soluble matriptases appeared to have little proteolytic activity. Treatment of stomach and colon cancer cell lines with epidermal growth factor stimulated the release of matripatase/HAI-1 complexes. In cancer cell lines secreting active trypsin, however, matriptase was released mostly as an inhibitor-free, two-chain active form. Trypsin seemed to activate the membrane-bound, latent matriptase on the cell surface. These results suggest that matriptase and trypsin cooperatively function for extracellular proteolysis.     

Inhibitory action of a new lectin from Xerocomus chrysenteron on cell-substrate adhesion

Lectins are carbohydrate-binding proteins which potentially link to cell surface glycoconjugates and affect cell proliferation. We investigated the effect of a new lectin from the mushroom Xerocomus chrysenteron (XCL) on cell proliferation using adherent and suspension cell lines. XCL caused a dose-dependent inhibition of proliferation of the adherent cell lines NIH-3T3 and HeLa. Several experiments suggest that disruption of cell-substrate adhesion is the main factor affecting cell growth inhibition. (i) No antiproliferative effect was observed on the SF9 cell line, which does not require to be attached to grow. (ii) XCL was shown to affect the adherence of cells following their suspension by trypsin treatment. (iii) XCL was localized on the cell surface where it would act as a coating agent. (iv) XCL induced morphological changes from well spread to rounded cells and disrupted the actin cytoskeleton. By contrast, flow cytometric analysis showed that XCL does not interfere with the cell cycle, and does not induce apoptosis.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

大鼠肝癌细胞系的体外培养、纯化及辐照效应

用二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌,取肝癌白色结节,０．２５％胰酶消化,ＲＰＭＩ—１６４０培养基建系成功,共获１３系肝癌细胞系,经反复传代（１０代以上）,细胞系均以肝实质瘤细胞和胆管上皮癌细胞为主,另外夹杂少量成纤维细胞。用ＰＨ７．２的ＰＢＳ冲洗、７５％酒精消毒流式细胞仪（ＦＣＭ）后,纯化该细胞来．收集肝实质癌细胞系,用６０Ｃｏγ射线辐照,剂量为８００Ｒａｄ和１０００Ｒａｄ,结果８００Ｒａｄ剂量使肝实质癌细胞生长受到明显抑制,细胞传代时间延长一倍,１０００Ｒａｄ剂量使细胞生长停止,不能再行传代。     

A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells.

The glycosaminoglycans (GAG) of human cultured normal glial and malignant glioma cell lines were studied using 35S-sulphate or 3H-glucosamine as markers. 35S-labelled GAG were assayed by precipitation with cetylpyridinium chloride; 3H-labelled sulphated GAG and 3H-labelled hyaluronic acid were quantitated after separation on a DEAE-cellulos column. The net production of GAG and the distribution, composition and turnover of GAG were similar in all of the normal cell lines tested, but showed a great variability in the malignant cell lines. Most of the glioma cell lines produced more hyaluronic acid and less sulphated GAG than the normal cell lines, but exceptions were noted. The GAG of the trypsin susceptible (pericellular pool of normal glial cells consisted mainly of heparan sulphate with only minor amounts of other GAG. The analogous material of most glioma cells showed hyaluronic acid as the major GAG. Material liberated by trypsin from EDTA-detached cells (membrane fraction) was enriched in heparan sulphate as compared to the entire pericellular pool. Substrate attached material (SAM) left with the plastic dish after EDTA treatment of normal cultures was rich in heparan sulphate, whereas SAM of glioma cells lacked heparan sulphate or showed greatly reduced amounts of this component. Release of newly synthesized GAG to the extracellular medium was a rapid process in the normal cells but was more or less delayed in the glioma cells. The extracellular medium of the malignant glioma cultures was consistently poor in dermatan sulphate, as compared to that of normal cultures.     

Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.     

5个品系小鼠胚胎干细胞系建立的方法学比较

以70％的大鼠心脏细胞条件培养基(RH-CM)为培养液,以小鼠胚胎成纤维细胞(PMEF)为饲养层,采用添加1％鸡血清的消化液和“连续离散法”作为小鼠Es细胞建系的改进方法,比较了5个品系小鼠ES细胞系建立的特点。与常规方法相比,3个近交系小鼠129／ter、C57BL/6J、BALB／c的ES细胞建系率分别由11．8％、3．7％和2．9％提高到33．3％、13．3％和19．4％,差异十分显著;直接采用改进的方法建立KM和ICR小鼠ES细胞系,建系率分别达12％和42．1％。讨论了ICM增殖的时间,即离散时机对ES集落形成及建系率的影响,结果显示：129／ter、C57BL/6J、BALB／c、KM和ICR小鼠品系ICM适宜的离散时机分别为增殖4～6d、3～3．5d、4d、4～5d和4～5d;同时,讨论了不同ES细胞建系所需最适宜的消化液浓度,其中BALB／c小鼠的ES细胞对高浓度的消化液十分敏感,0．05％Trypsin-0．008％EDTA是其比较理想的离散浓度。设计了两种离散方法,即“一次离散法”和“连续离散法”,用来离散增殖的ICM和ICM离散后出现的ES集落,结果表明：后者在建系过程中的作用明显优于前者。RH-CM与添加uF的常规ES细胞培养基相比,不但具有显著抑制小鼠ES细胞分化、维持其二倍体核型的作用,而且明显促进ES细胞的贴壁生长。新建细胞系鉴定结果表明,这一改进方法有效地维持了其作为多能性胚胎干细胞的一系列特征。     

Partial purification of a mast cell growth-enhancing activity and its separation from IL-3 and IL-4

A growth factor acting synergistically with IL-3 on thiol-sensitive "mucosal type" bone marrow-derived mast cell lines, and therefore termed mast cell growth enhancing activity, is present in PWM stimulated spleen cell conditioned medium. Mast cell growth enhancing activity can be partially purified and completely separated from IL-3, IL-4, and IL-5, and for the most part from IL-6 and GM-CSF using strong cation exchange and Procion red affinity chromatography. Mast cell growth enhancing activity binds to Con A-Sepharose and can be digested with trypsin and chymotrypsin. It shows a Mr ranging from 37 to 43 kDa under nonreducing SDS-PAGE and a main isoelectric point ranging from 6.2 to 7.3.     

Trypsin modifies the activity of adenylate cyclase from normal, malignant and hybrid mammalian cells

Adenylate cyclase (ATP pyrophosphate-lyase (cylizing), EC 4.6.1.1) activity, measured in homogenates of normal, malignant and hybrid mammalian cell lines, is enhanced and subsequently inhibited by increasing concentrations of trypsin (EC 3.4.21.4). Treatment of intact cells with trypsin appears to cause latent activation of adenylate cyclase (i.e. activation which is only expressed after homogenization of the cells). Conversely, adenylate cyclase activity of a normal Chinese hamster fibroblast cell line is inhibited in intact cells by trypsin through the degradation of some site on the outer surface of the plasma membrane. The prostaglandin E1 receptor is not affected by trypsinization of cells.     

A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry

High-throughput flow cytometry of adherent cells is difficult because the creation of single cell suspensions can damage cells and yield artificial results. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines (Panc-1 and AsPC-1) to produce alterations in the cell cycle and intracellular protein expression. The cells in 96-well plates were disassociated using a collagenase and trypsin mixture. Fluorescence-activated high-throughput flow cytometry evaluated cellular viability as well as surface and intracellular protein expression. Cell cycle analysis was performed using 7-aminoactinomycin D and intracellular protein characterization was performed using a fluorescein-labeled monoclonal antibody against activated caspase-3. The collagenase–trypsin-based protocol increased single cell events from 31.9 ± 0.5% using trypsin alone (standard) to a range of 62.1% to 85.5% without adversely affecting viability. High-throughput flow cytometry demonstrated that the addition of collagenase to the disassociation solution not only permitted significantly higher rates of single cell creation, but it did not negatively affect the doxorubicin-induced protein expression. This protocol allows for expedient and effective disassociation of adherent human cells in order to investigate alterations in specific cellular enzymes and pathways.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

Proteinase-activated receptor-2 expression in breast cancer and the role of trypsin on growth and metabolism of breast cancer cell line MDA MB-231

Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot. Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors.