Ohtsura Niwa

Unstable Expression of E‐Cadherin Adhesion Molecules in Metastatic Ovarian Tumor Cells

E‐Cadherin is a member of the cadherin family, which plays a key role in intercellular adhesion in various tumors as well as in normal tissues. Here, we examined the expression of this adhesion molecule in a murine ovarian tumor line OV2944, whose sublines show different degrees of spontaneous metastasis from subcutaneous sites; sublines LM‐1 and LM‐3 exhibit a low metastatic activity but a variant subline HM‐1 has a high metastatic activity. When the expression of E‐cadherin in these cells was examined by immunoblot analysis, the highly metastatic HM‐1 cells was found to express an extremely small amount of this molecule, as compared with a high level of E‐cadherin expression in the weakly metastatic LM‐1 and LM‐3 cells. Northern blot analysis showed that the amount of tanscripts from the E‐cadherin gene is proportional to the amount of proteins detected in these cells. Immunofluorescence staining revealed that cells of the highly metastatic line were heterogeneous, that is, their cultures contained both E‐cadherin‐positive and negative cells. In contrast, cells of the weakly metastatic lines homogeneously expressed E‐cadherin. When the highly metastatic line was subcloned, all the subclones consisted of E‐cadherin‐positive and negative cells. These results suggest that the expression of E‐cadherin gene is not stably controlled in the highly metastatic line.

Embryonal long terminal repeat-binding protein is a murine homolog of FTZ-F1, a member of the steroid receptor superfamily.

The embryonal long terminal repeat-binding protein, ELP, is present in undifferentiated mouse embryonal carcinoma cells. It binds to and suppresses transcription of the Moloney leukemia virus long terminal repeat in undifferentiated murine embryonal carcinoma cells. We report here that ELP is a mouse homolog of Drosophila FTZ-F1, which positively regulates transcription of the fushi tarazu gene in blastoderm-stage embryos of the fly. As members of the steroid receptor superfamily, ELP and FTZ-F1 have both DNA binding and putative ligand binding domains which are well conserved between the two. ELP and FTZ-F1 function in cells in the extremely early stage of development. A high degree of conservation between the two transcription factors during the evolution of these species indicates the importance of their functions in early-stage embryogenesis. In addition, the sequence elements they recognize do not contain repeat units, in contrast to other steroid receptors, which usually bind to either palindromic or direct repeat sequences.

Mechanism of suppression of the long terminal repeat of Moloney leukemia virus in mouse embryonal carcinoma cells.

Sequence-specific DNA-binding proteins that bind to the long terminal repeat (LTR) of Moloney leukemia virus in undifferentiated and differentiated mouse embryonal carcinoma (EC) cells were identified by gel retardation assay. The proteins that bind to the CCAAT box were present in both undifferentiated and differentiated EC cells. The amounts and the number of species of the proteins that bind to the enhancer and the GC-rich region were far lower in undifferentiated EC cells than in the differentiated counterparts. These proteins were supposed to be transcriptional activators. Proteins that bind upstream of the enhancer, namely, the -352 to -346 region and the -407 to -404 region, were identified. These proteins were designated the embryonic LTR-binding protein (ELP) and the LTR-binding protein, respectively. The ELP was present only in undifferentiated EC cell lines. The LTR-binding protein was detected in all cell lines tested. The mechanism of suppression of the LTR was investigated by the chloramphenicol acetyltransferase assay. The enhancer and the GC-rich region of the LTR functioned poorly in undifferentiated cells. When eight copies of ELP-binding sequences were inserted upstream of the enhancer region, expression of the chloramphenicol acetyltransferase gene was reduced about threefold in ECA2 cells. From these data, we concluded that two mechanisms, the shortage of activator proteins and the presence of a negative regulatory protein (ELP), are involved in the suppression of the LTR in undifferentiated EC cells.

Induction of Transplantable Tumors by Repeated Subcutaneous Injections of Natural and Synthetic Vitamin E in Mice and Rats

Natural vitamin E and synthetic vitamin E (dl/‐α‐tocopheryl acetate) were tested for their tnmorigenicity in rodents. Transplantable tumors, at the site of injection, were induced by repeated injections of these compounds in two strains of mice, NFS/N and C57BL/6N × C3H/He Fl, and in a strain of rats, Fischer 344. Natural vitamin E was tumorigenic in both strains of female mice only when injected with soya oil. In contrast, dl‐α‐tocopheryl acetate alone was capable of inducing tumors in Fischer 344 rats. Only one out of 5 male NFS/N mice given dl‐α‐tocopheryl acetate developed a tumor. Therefore, Fischer 344 rats were more susceptible to tumor formation by dl‐α‐tocopheryl acetate than NFS/N mice. dl‐α‐Tocopheryl acetate with soya oil or with palm oil also resulted in the formation of transplantable tumors in NFS/N mice and Fischer 344 rats. There was no difference in the tumor incidence between mice treated with dl‐α‐a‐tocopheryl acetate alone and dl‐α‐tocopheryl acetate plus soya oil or palm oil. However, in rats, the incidence was lower for a group treated with dl‐α‐tocopheryl acetate plus palm oil than for those with dl‐αa‐tocopheryl acetate alone and with dl‐α‐tocopheryl acetate plus soya oil.

Overexpression and Amplification of the c‐myc Gene in Mouse Tumors Induced by Chemicals and Radiations

We examined expression of the c‐myc gene by the dot blot hybridization of total cellular RNA from mouse primary tumors induced by chemicals and radiations. Expression of the c‐myc gene was found to be elevated in 69 cases among 177 independently induced tumors of 12 different types. DNA from tumors overexpressing the myc gene was analyzed by Southern blotting. No case of rearrangement was detected. However, amplification of the c‐myc gene was found in 7 cases of primary sarcomas. These included 4 cases out of 24 methylcholanthrene‐induced sarcomas and 3 cases out of 7 α‐tocopherol‐indnced sarcomas. We also analyzed 8 cases of sarcomas induced by radiations, but could not find changes in the gene structure of the c‐myc gene. Thus, our data indicate tumor type specificity and agent specificity of c‐myc gene amplification.

Over-expressed ZF5 Gene Product, a c-myc-binding Protein Related to GL1-Kruppel Protein, Has a Growth-suppressive Activity in Mouse Cell Lines

ZF5 encodes a zinc finger protein, which contains five C2H2‐type zinc fingers showing homology with the zinc finger of the Kruppel family, and binds to two sites in the mouse c‐myc promoter. We report the effect of over‐expression of ZF5 on cell growth. ZnCl2 treatment suppressed the growth of a mouse fibroblast cell line (L cells) transfected with the wild‐type ZF5 gene driven by the metallothionein promoter. Cells transfected with the wild‐type ZF5 gene formed colonies two‐ to fivefold less efficiently than those transfected with the mutant ZF5 gene in P19, NIH3T3, 3T3‐L1 and L cells. Over‐expression of ZF5 did not cause c‐myc down‐regulation or arrest of the cell cycle, but increased the DNA content.

Somatic Mutation during Metastasis of a Mouse Fibrosarcoma Line Detected by DNA Fingerprint Analysis

Metastatic nodules were examined by DNA fingerprint analysis. The probes used, Pc‐1 and Pc‐2, detect mutations as shifts in bands of the minisatellite loci which are dispersed among chromosomes. Four clonal lines of a fibrosarcoma from an F1 mouse (C57BL/Ka × C3H/He) were selected for various metastatic potentials upon inoculation into syngeneic mice. These four lines exhibited many extra bands resulting from recombination and/or DNA slippage, indicating accumulation of mutations during the successive passages in mice. One of the four, a 505 cell line which had been passaged extensively in vitro and consisted of a heterogenous population, was inoculated into thirteen syngeneic mice, and gave rise to six lung metastatic nodules in two mice. All the nodules showed band‐patterns distinct from one another, although nodules within a given mouse tended to show similar patterns. When a genetically tagged 505‐05‐01 clone was analyzed, three of nine metastatic nodules obtained also revealed new bands. These results strongly suggest that somatic mutations occur at a high frequency during metastasis, providing direct evidence of genetic instability of the tumor cells.

Analysis of clonal evolution in a tumor consisting of pSV2neo-transfected mouse fibrosarcoma clones

The process of clonal evolution was analyzed in a line of methylcholanthrene‐induced mouse fibrosarcomas. The tumor cells were transfected with pSV2neo gene and 22 clones were randomly isolated. Genetically tagged clones were mixed and inoculated into syngeneic mice. Southern blot analysis revealed that one of the clones, no. 11, dominated both in tumors in situ and in lung metastatic nodules. No. 11 clone and other clones were similar in growth rates in vitro and in vivo, in spontaneous and experimental metastatic abilities, in immunogenicity, and in the capacity of intercellular communication in vitro. Although no. 11 clone overgrew other clones in vivo, this was not the case when clones were mixed and maintained in vitro. We conclude that clonal interactions in vivo may be responsible for the dominance of no. 11 clone in the tumor. It is likely that the preferential metastasis of no. 11 clone to the lung may be a simple reflection of the proliferative advantage of the dominant clone in the tumor in situ.

A Defect in Cell–to–cell Adhesion via Integrin–Fibronectin Interactions in a Highly Metastatic Tumor Cell Line

We investigated the role of integrin–fibronectin (FN) interactions in tumor cell adhesion. Two cloned tumor cell lines designated OV–LM (low–metastatic) and OV–HM (high–metastatic) were isolated from a murine ovarian carcinoma, OV2944. OV–LM and OV–HM cells exhibited high and low RGDS–sequence–dependent adhesiveness to FN, respectively. Both lines expressed comparable levels of α5 and αv integrins, which are capable of reacting with RGDS on FN. To compare the functions of these integrins between the two tumor lines, the signaling mechanism following FN stimulation was examined. Significant levels of phosphorylation of focal adhesion kinase(FAK)were detected in bothOV–LM and OV–HM cells before FN stimulation. Whereas the level of FAK phosphorylation was appreciably enhanced in OV–LM cells stimulated with FN, stimulation of OV–HM cells with FN induced a reduction in the FAK phosphorylation in association with a significant decrease in theamount of FAK protein in the soluble compartment of cell lysates. A difference in the deposition of FN on the cell surfacewas also observed between the two types of tumor lines; OV–HM cells had an appreciably smaller amount of FN than OV–LM. Consistent with the functional abnormality of the integrin–FAK system and the smaller amount of FN on OV–HM, this clone exhibited a reduced cell–cell adhesion in the in vitro cell aggregation assay. Namely, OV–LM cells displayed a time–dependent increase in the formation of cell aggregates, whereas most OV–HM cells remained single. The formation of aggregates by OV–LM cells was inhibited by addition of RGDS peptide. These results indicate that the highly metastatic clone, OV–HM, exhibits a decreased capacity of cell–cell adhesion mediated by integrin–FN interactions and suggest that this defect is mainly due to the dysfunction of integrins/FAK rather than a decrease in the amount of integrins expressed on tumor cells.

Myeloproliferative disorder due to abnormal production of hematopoietic stimulators.

A new kind of myeloproliferative disorder (L-8313) has been discovered. It was transplantable into syngeneic mice with spleen cells. The mice showed hepato-splenomegaly with a marked leukocytosis and anemia 3 weeks after transplantation of L-8313 cells. The number of GM-CFU and CFU-S per spleen increased to more than 40 times normal. The results of chromosomal and PGK analysis demonstrated that these increased stem cells were of host origin. Both the culture medium of the spleen cells and the serum from L-8313 bearing mice showed high levels of IL-3, BPA and CSF. Consequently, hematopoietic cells of the host mice underwent remarkable proliferation in response to these stimulating factors when L-8313 cells were transplanted. We also have been successful in establishing an in-vitro cell line and have maintained it for over one year. The phenotype of L-8313 cells was Thy 1.2 positive. Some L-8313 cells showed a positive acid phosphatase reaction but the cytochemical character of myeloid lineage was not observed. Therefore, L-8313 is considered to be a T-cell derived hematopoietic regulatory cell neoplasm with the ability to produce several hematopoietic stimulating factors.