N.J. de Both

E-cadherin gene mutations are rare in adenocarcinomas of the oesophagus.

SummaryReduced expression of E-cadherin, a cell–cell adhesion molecule, is observed in oesophageal adenocarcinomas and correlates with less favourable pathological parameters and survival. To determine if genetic events lead to reduced E-cadherin expression in these patients, we screened all 16 exons of the E-cadherin gene for mutations with the polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) technique in 49 resection specimens, including four loco-regional lymph node metastases, four established cell lines and four xenografts. Fifteen exon-spanning primer pairs were used, and in nine amplicons aberrant bands were detected. Sequencing of the amplicons revealed a one base-pair deletion (codon 120; exon 3) in cell lines JROECL 47 and JROECL 50 leading to a premature downstream stop codon. Polymorphisms were identified for amplicons 1, 4/5, 11, 12, 13, 14 and 16 corresponding with data from the literature. Three new polymorphisms were detected for amplicons 2, 3 and 4/5. Loss of heterozygosity (LOH) of the E-cadherin locus on 16q22.1 was examined with four polymorphic markers. LOH was found in 31 of the 48 informative cases (65%). These results show that, despite the frequent LOH of the E-cadherin locus, mutations in the E-cadherin gene are rare events and can not be held responsible for down-regulation of E-cadherin observed in the majority of adenocarcinomas of the oesophagus.

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat: A study of enzymes bound to subcellular organelles

Abstract The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus. The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R). Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β- N -acetylglucosaminidase) are not affected by changing crypt cell kinetics. Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.

A comparative evaluation of various invasion assays testing colon carcinoma cell lines

SummaryVarious colon carcinoma cell lines were tested in different invasion assays, i.e. invasion into Matrigel, into confluent fibroblast layers and into chicken heart tissue. Furthermore, invasive capacity and metastatic potential were determined in nude mice. The colon carcinoma cells used were the human cell lines Caco-2, SW-480, SW-620 and HT-29, and the murine lines Colon-26 and -38. None of the human colon carcinoma cells migrated through porous membranes coated with Matrigel; of the murine lines, only Colon-26 did. When incubated in a mixture of Matrigel and culture medium non-invading cells formed spheroid cultures, whereas invading cells showed a stellate outgrowth. Only the heterogeneously shaped (epithelioid and stellate) cells of SW-480 and SW-620 and the spindle-shaped cells of Colon-26 invaded clearly confluent skin and colon fibroblasts as well as chicken heart tissue. However, when transplanted into the caecum of nude and syngeneic mice, all the lines tested were invasive with the exception of Caco-2 cells. We conclude that the outcome of in vitro tests measuring the invasive capacity of neoplastic cells is largely dependent on the test system used. Invasive capacity in vitro is strongly correlated with cells having a spindle cell shape, vimentin expression and E-cadherin down regulation. In contrast, HT-29 and Colon-38 cells having an epithelioid phenotype were clearly invasive and metastatic in vivo, but not in vitro.

DMSO-induced terminal differentiation and trisomy 15 in myeloid cell line transformed by the Rauscher murine leukemia virus

A new myeloid cell line was isolated from a myeloid leukemia obtained after infection of BALB/c mice with Rauscher murine leukemia virus (R-MuLV). After syngeneic transplantation of leukemic cells tumor formation was induced. Of one of these tumors a permanent cell line could be established. The cells grow in suspension culture with a doubling time of 18 h and morphologically and cytochemically show all the characteristics of myelocytes. The cells carry trisomy of chromosome 15. These cells prove to be completely independent of colony stimulating activity (CSA) regarding both their growth and their differentiation capacity. One of the main characteristics of this cell line is its inducibility for terminal differentiation after treatment with dimethylsulfoxide varying in concentrations from 0.5% to 1.5%. After two days metamyelocytes and after three to four days granulocytes and macrophages formed. The differentiation of these cells goes together with an increase of lysosomal enzyme activities like β-N-acetylglucosaminidase and lysozyme.

The expression of differentiation antigens by Rauscher virus-induced erythroid, lymphoid and myeloid cell lines.

Rauscher murine leukemia virus-induced erythroid, lymphoid and myeloid cell lines were characterized with respect to the expression of differentiation antigens using a panel of monoclonal antibodies. The expression of differentiation antigens was measured in a two-step micro ELISA procedure. The cell lines express a number of early markers and lack a number of mature markers characteristic for the respective cell lineages. Moreover they express a number of surface markers which are not or only rarely found on their normal counterparts. The expression of differentiation antigens indicates that the cell lines investigated are arrested in an immature stage of differentiation. This observation implies that the Rauscher virus preferentially transforms early hemopoietic cells.

Nucleotide sequence of the Rauscher murine leukaemia virus long terminal repeat.

The long terminal repeat (LTR) of Rauscher murine leukaemia virus (MuLV) has been sequenced. It differs in only three positions from the LTR of Rauscher spleen focus-forming virus (SFFV), and in four positions from the LTR of Rauscher mink cell focus-inducing virus (MCFV). It is unlikely that these differences account for differences in leukaemogenicity or tissue tropism of Rauscher MuLV, SFFV and MCFV. In contrast to the LTR of Friend MuLV, the Rauscher MuLV LTR contains only one copy of a tandem direct repeat. This repeat includes an enhancer core sequence.

Oncogene expression in rauscher murine leukemia virus induced erythroid myeloid and lymphoid cell lines

A comparative study on the expression of nuclear and cytoplasmic oncogenes was carried out using the Northern blotting technique, in Rauscher virus induced primary leukemias and the more malignant transformed cell lines derived from them. The latter grow permanently in vitro. Hyperplastic spleens obtained from mice recovering from anemia were analysed as controls. In addition to the detection of mRNAs, Southern blotting was carried out to observe whether rearrangement or amplification of oncogenes had occurred. The results show that the nuclear oncogenes c-myc, c-myb and p53 are strongly expressed in leukemic tissue, whereas c-fos transcripts show a much weaker hybridization. The expression of two of these oncogenes, c-myc and c-myb was followed during differentiation in myeloid leukemic cells and showed a gradual decrease when compared with the actin gene, which is constitutively transcribed. A large number of cytoplasmic oncogenes is expressed in the leukemic cells lines, i.e. c-abl, c-fms, c-fes, c-src, c-ros, c-H-ras, c-K-ras and N-ras. Of these, transcripts coding for c-abl and c-src were absent in blast cells of acute erythroid leukemias. Transcripts coding for c-erb, c-mos and c-sis could also not be detected. A number of putative oncogenes which are reported to play a role in Moloney and Friend virus induced leukemias for instance pim-1, fis-1, fim-1 and fim-2 were also used for screening. Only expression of pim-1 in Rauscher virus induced myeloid leukemic cells and in primary acute erythroid leukemias could be observed. At the DNA level no rearrangement or amplification of any of the oncogenes investigated could be detected. The results show that a number of oncogenes are expressed simultaneously in the same leukemic tissue or cell lines. It therefore seems likely that the presence of transcripts of different oncogenes is associated with the progression of leukemia, but is not the primary cause of leukemogenesis or of the transformation of these cells into established cell lines.

Factors influencing antibody-mediated cytotoxicity during the immunotherapy of Rauscher-virus-induced myeloid leukemic cells

SummaryThe present study was undertaken to determine the factors that influence antibody-mediated cytotoxicity during immunotherapy of virally transformed tumor cells. As model a Rauscher-virus-induced myeloid leukemic cell line of BALB/c origin (RMB-1) was used, which forms disseminated tumors, when inoculated intravenously in BALB/c mice. As previously reported, prolonged survival was obtained when tumor-bearing mice were treated in vivo with a single high dose of a tumor-specific IgG2a monoclonal antibody. This study shows that antibody-dependent cellular cytotoxicity is an important mechanism involved in tumor cell destruction. Since in vitro studies showed that peritoneal macrophages were capable of killing RMB-1 cells in the presence of tumor-specific monoclonal antibody and since in the tumors of mice treated with monoclonal antibody a high influx of macrophages was observed histologically, it is likely that macrophages play an important effector role in elimination of tumor cells. Successful therapy in C5-complement-deficient tumor-bearing mice suggests that complement-dependent cytotoxicity does not play a major role. In nude (T-cell-deficient) mice the therapeutic effect of tumor-specific IgG2a antibody was significantly less than in immunocompetent mice. Although infiltration analysis of tumors of treated and untreated mice showed equally low numbers of helper-T and suppressor/cytotoxic T-cells, the mortality studies of T-cell-deficient and immunocompetent mice indicate that T-cells play a substantial, auxillary role during antibody-mediated, tumor destruction in our model.

Effects of Rauscher murine leukemia virus on hemopoietic organ-derived clonogenic fibroblasts

Fibroblast colony-forming units (CFU-F) in bone marrow and spleen were investigated after infection of BALB/c mice with the Rauscher leukemia virus complex (RLV) or with cellularly cloned helper virus (R-MuLV). Both viral preparations induced a transient suppression of CFU-F prior to the development of leukemia. A second CFU-F decrease was observed in the terminal phase of R-MuLV-induced chronic lymphoid or myeloid leukemias, but not in mice with a RLV-induced acute erythroleukemia, which are highly viremic. When a number of Rauscher virus transformed leukemic cell lines were injected intravenously an erythroid and a T-cell line suppressed CFU-F values, but a third non-virus producing B-cell line did not. In contrast to the in-vitro situation, in-vitro inhibition of CFU-F was only observed in conditioned medium of an erythroid cell line with undetectable reverse transcriptase activity, whereas conditioned medium of lymphoid and myeloid cell lines were not inhibitory irrespective of reverse transcriptase activity. Together these results indicate that murine leukemia viruses can suppress the numbers of detectable CFU-F in vivo in an early phase of the disease and that leukemic cells may inhibit CFU-F proliferation by a mechanism which is not related to viremic state of the animal or the production of virus by these cells.

Decreased tumorigenicity of Chinese hamster cells after fusion with tumorigenic mouse myeloid leukemia cells

Hybrid cell lines, derived after fusion of tumorigenic mouse and Chinese hamster cells, were assayed for their tumor forming capacity in nude mice. Partial suppression of tumorigenicity was observed in some of the hybrids tested, suggesting complementation for tumorigenicity.