Cell surface microvilli and cell agglutinability.

Detached cells of some transformed mouse fibroblast lines have a villous surface whereas similarly treated cells of other lines are relatively smooth. These differences in surface morphology of detached cells are not reflected in their agglutinability with ConA and they cannot unambigously be explained from their morphology in situ. Treatments of normal and transformed Swiss mouse fibroblasts that induce marked changes in agglutinability with ConA do not cause equivalent changes in surface morphology. It is, therefore, unlikely that agglutinability of mouse fibroblasts by ConA is determined by the number of microvilli on the cell surface.     

Cell-surface and secreted proteinases and guanidinobenzoatase activity

Hydrolysis of p-nitrophenyl guanidinobenzoate is characteristic of a tumour cell surface proteinase. We found this activity in three transformed or tumour cell lines, but not in two normal fibroblast strains. Immunochemical inhibition of plasminogen activator activity did not affect guanidinobenzoatase activity.     

Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins (“delRGD” or “RGE”) supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.     

In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells

The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.     

Transformation of hamster embryonic fibroblast cells by UV-irradiated human cytomegalovirus.

Infection of hamster embryonic fibroblast (HEF) cells with UV-irradiated human CMV resulted in appearance of transformed clones. The established cell line (87-TRH-5) when inoculated subcutaneously induced tumour in newborn hamsters. The 87-TRH-5 cell line and the cell lines (87-TRH-5-Tsc1, 87-TRH-5-Tsc2, 87TRH-5-Tsc3) developed from the induced tumour, proved to bear CMV-spcific cytoplasmic and surface antigens. Demonstration of infectious CMV or MCV-specific nuclear antigen in 87-THR-5-cells has failed.     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Development of normal and genetically dystrophic mouse muscle in tissue culture : I. Prefusion and fusion activities of muscle cells: Phase contrast and time lapse study

Dispersed cell cultures, derived from the forelimbs and hindlimbs of genetically dystrophic (dy/dy) and normal (+/+) day mouse embryos were studied with phase contrast microscopy and time lapse cinematography. The composition of the cell populations, and the prefusion and fusion activities of the cells were analysed. Forelimbs of both normal and dystrophic embryos consistently yielded fewer mononucleated cells, more fat cells and fewer myoblasts than hindlimbs, but there was no difference in the population of cells from normal and dystrophic limbs. During prefusion, myoblasts (both normal and dystrophic) exhibited (1) an apparent lack of contact inhibition of locomotion, which was in actuality an extensive movement of one myoblast under another; (2) formation of prefusion aggregates that broke up and realigned into new aggregates before fusion; (3) a special type of post-mitotic association and reassociation, not found among fibroblasts. Onset of rapid cell fusion of myoblasts occurred in a 4 to 8 h period, and was directly dependent upon initial cell concentration. No differences were found between cultures of normal and dystrophic cells in their prefusion activities or in time of onset of rapid cell fusion, when initial concentrations of cells were kept constant. The results of the present study are compared with those of other in vitro studies of dystrophic muscle.     

Analysis of changes in natural cytostatic and cytotoxic activity of mouse spleen cells after treatment with cyclophosphamide and alpha-interferon

Cytostatic and cytotoxic activity of mouse spleen cells against normal and tumour target cells has been studied. The comparative analysis of mouse spleen cell cytostatic and cytotoxic activity after exposure to cyclophosphamide has shown that the effectors of natural cytotoxic activity are highly sensitive to cyclophosphamide, while cytostatic effectors are heterogeneous in their sensitivity to cyclophosphamide. Pretreatment of spleen cells with alpha-interferon produced an increase in cytotoxic and cytostatic activity against tumour target cells. The cells of lymphoid organs (spleen, bone marrow, thymus) had greater distinctions in cytotoxic than in cytostatic activity against tumour target cells.     

Further investigation of the increased transfer ribonucleic acid methylase activity in tumours of the mouse colon

1. Extracts prepared from tumours of the mouse colon induced by 1,2-dimethylhydrazine were considerably more active in catalysing the methylation of tRNA than were extracts from normal colon. The enhanced activity was observed when both unfractionated ;methyl-deficient' tRNA and purified tRNA preparations from yeast and bacteria were used as substrates for methylation. 2. The methylated bases produced in these reactions were identified. There were no differences between the products of the reaction catalysed by extracts of tumour and normal colon. 3. The increased activity of tRNA methylases was not due to the presence in the extracts of stimulatory or inhibitory molecules of low molecular weight such as polyamines or S-adenosylhomocysteine. 4. Other enzymes concerned with tRNA metabolism (RNA polymerase, ATP-tRNA adenylyltransferase, aminoacyl-tRNA ligases) were also increased in activity in the tumour tissue. 5. The extent of methylation of a limiting amount of tRNA was greater when tumour extracts were compared with controls, but in no case was it possible to achieve a stoicheiometric methylation of the purified tRNA preparations used as substrates, and the tumour extracts were not able to methylate tRNA obtained from normal mouse colon. We conclude that the tumours contained greater activities of tRNA methylases but that there was no evidence for changes in the specificity of these enzymes during neoplastic growth.     

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.     

Functional consequences of haem orientational disorder in sperm-whale and yellow-fin-tuna myoglobins.

With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

Disruption of the gene encoding the ubiquitin-conjugating enzyme UbcM4 has no effect on proliferation and in vitro differentiation of mouse embryonic stem cells

The ubiquitin-conjugating enzyme UbcM4, which is identical to the human enzyme UbcH7, was previously shown to be essential for normal mouse development. In order to study the possible role of UbcM4 for cell proliferation and in vitro differentiation, we here describe the establishment and characterization of fibroblast and embryonic stem cell lines with partial or complete inactivation of the UbcM4 gene. ES cell lines in which both alleles of the gene were inactivated by targeted mutagenesis showed no differences in growth rates, cell cycle progression and in vitro differentiation when compared to wild-type ES cells. Fibroblast cell lines with a partially inactivated UbcM4 gene were derived from embryos of the previously described A6 mouse mutant, where retrovirus integration has resulted in a recessive lethal mutation. As in the mutant embryos, steady levels of RNA and protein in the cell lines were reduced by about 70%. The mutant cell lines showed no differences in immortalization kinetics, growth rates and cell cycle progression when compared to wild-type fibroblasts. Taken together, our results strongly suggest that UbcM4-mediated ubiquitination and degradation are not necessary for proteins involved in the maintenance and growth of cells.     

Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium

Fibroblasts are a diverse cell type and display clear topographic differentiation and positional memory. In a screen for fibroblast specific markers we have characterized four monoclonal antibodies to endosialin (TEM1/CD248). Previous studies have reported that endosialin is a tumour endothelium marker and is localized intracellularly. We demonstrate conclusively that endosialin is a cell surface glycoprotein and is predominantly expressed by fibroblasts and a subset of pericytes associated with tumour vessels but not by tumour endothelium. These novel antibodies will facilitate the isolation and classification of fibroblast and pericyte lineages as well as the further functional analysis of endosialin.     

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

Anti‐angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion‐associated domain in MUC4 and other proteins (AMOP) domain at the C‐terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C‐terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)‐basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF‐stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase‐dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen‐tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.     

抗人肺癌细胞单克隆抗体的制备及其特性的研究

用人肺鳞癌细胞LTEP-78细胞系免疫Blab/c小鼠获得3株抗人肺癌细胞的单克隆抗体杂交瘤系。其中BLTI-01株经六次克隆化培养,体外传代8个月以上。BLTI-01与白细胞抗原及血型抗原基本上无交叉反应;与骨髓细胞无交叉反应;与癌胚抗原和胎甲球蛋白不相关;与肺鳞癌、肺腺癌细胞系及部分其它肿瘤细胞呈阳性反应。     

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.     

Fine structure of submandibular glands of mice with testicular feminization (Tfm/Y)

Summary The fine structure of the submandibular gland of the mouse with testicular feminization (Tfm/Y) was studied by light and electron microscopy. The architecture of the Tfm/Y gland proved to be rather similar to that of the normal female mouse in both tubular ratio and structure. Granular convoluted tubular cells in Tfm/Y mice characteristically had fewer secretory granules and increased cytoplasmic vacuoles than normal littermates, suggesting an altered synthesis of secretory granules in this cell type of the Tfm/Y mouse. Moreover, there were differences in the ultrastructure of submandibular glands between Tfm/Y and normal female mice. In the gland of the Tfm/Y mouse, basal striations of the striated secretory tubular cells were not so developed and granular intercalated duct cells were less than those of normal females. These findings support the evidence that the secretory tubule of the mouse submandibular gland responds to androgens, resulting in accentuated development in the male, while also suggesting the possibility that the mouse submandibular gland is regulated by other factors which lead to the prominent sexual dimorphism observed in this gland.