Transplantation of axolotl heads.

Favorable conditions for organ transplantation exist for some populations of European laboratory axolotls, making transplantations of heads possible. Survival of the transplants is prolonged because homograft reactivity of the host animals is absent. Heads transplanted to the backs of other axolotls grow rapidly and show many reactions characteristic of normal axolotl heads. The behavior of the transplants is independent of that of the host animals.

Trisomy 15 and other nonrandom chromosome changes in Rauscher murine leukemia virus-induced leukemia cell lines.

The cytogenetics of Rauscher murine leukemia virus-induced erythroid, myeloid, and lymphatic leukemias were studied in BALB/c and DBA/2 mice. In primary virus-induced leukemias, no chromosome abnormalities were found. However, in tumors derived from transplanted leukemia tissue and in cell lines obtained from these tumors, euploidy and mostly aneuploidy were observed that varied according to the type of tumor cells and the number of passages. Eleven erythroleukemia cell lines showed aneuploidy from the first in vivo transplant stage on. Nonrandom abnormalities were found: trisomy 15 in 9 of 11 lines, followed by trisomy 3 in 8 of 11 lines and monosomy 6 in 5 of 11 lines. Subsequent evolution of the karyotypes was frequent (10 of 11 lines) and rapid. In vivo cell lines established from these tumors showed many structural rearrangements. Three myeloid lines revealed a stable karyotype with no or only minor changes: trisomy 15 in 1 line and normal diploid in 2 lines. One lymphatic leukemia cell line established in vitro presented with a very stable karyotype: trisomies 14 and 15. Another in vivo transplantable line showed trisomies 11, 9, and 17. These results suggest that trisomy of chromosome 15 plays a significant role in tumors derived from different types of mouse leukemias.

Erythrocyte production and survival in Rauscher murine leukemia virus-infected BABL/c mice.

Red blood cell production in normal and Rauscher murine leukemia virus-infected mice was investigated using 55Fe as a marker. Using autoradiographic techniques, increases in the percentage of labeling of red blood cells were found in blood smears taken at different time intervals after pulse labeling of the erythroid precursor cells. The total reticulocyte production per unit time is more than 2.8-fold in Rauscher murine leukemia virus-infected mice as compared with that in uninfected mice. The life span of the newly formed cells was measured after [51Cr]chromate labeling of transfused erythrocytes in infected and in control mice. The life span was indicated by time that one-half of the labeled erythrocytes disappeared (t1/2) was reduced to one-quarter of that of erythrocytes of uninfected mice. The functioning of the newly formed cells was analyzed by measuring the glucose utilization versus lactate production and by measuring the activities of a number of enzymes involved in glucose metabolism. Comparison of glucose metabolism in normal and leukemic mice and in mice recovering from artificially induced anemia revealed that the metabolic activity of erythrocytes from leukemic mice corresponds to the activity of young erythrocyte populations. The increased reticulocyte production is, apparently, a result of the degree of anemia in infected animals. This anemia is not compensated for, however, since the loss of erythrocytes surpasses the flux of new red blood cells from the hematopoietic organs into the peripheral blood.

Spi-1 oncogene activation in Rauscher and Friend murine virus-induced acute erythroleukemias.

The Friend viruses, like the Rauscher virus, cause murine acute erythroleukemias which evolve in a similar multistep process. In previous studies it has been described that the late malignant proerythroblastic transformation induced by the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP) is correlated with Spi-1 oncogene activation by insertional mutagenesis. In this paper we report that Spi-1 genomic rearrangements were also observed in 90% of tumors induced by the anemia-inducing strain of Friend spleen focus-forming virus (SFFVA) and in all Rauscher-induced tumors analyzed. SFFVA and Rauscher proviral insertions occurred in the viral integration cluster previously characterized in SFFVP-induced tumors. The Spi-1 1.4-Kb messenger RNA was found highly expressed in all SFFVA and Rauscher-induced malignant cells as compared to normal tissues. The nucleotide sequence of Spi-1 cDNA isolated from a library constructed from SFFVA-induced tumor cells revealed no difference between the Spi-1 gene transcripts expressed in both SFFVP and SFFVA-induced leukemic cells. These results indicate that Spi-1 gene activation is a general feature in the malignant proerythroblastic transformation which occurs in mice infected with Friend and Rauscher viruses.

The effect of Rauscher murine leukemia virus infection on the hemopoietic system of BALB/c mice. Cell proliferation and cell loss.

Cell proliferation was investigated in normal and Rauscher Leukemia Virus-infected BALB/c mice. Five days after inoculation, islands of leukemic blasts arose in the red pulp, and proliferated as shown by autoradiographic analysis after a pulse of 3H-Thymidine. These cells subsequently infiltrated the whole spleen and 3 weeks after infection about 60% of the spleen consisted of large immature erythroblast-like cells. Repeated injections of 3H-Thymidine led to uniform labeling of 85% of the spleen cells. Cell cycle analysis showed that for bone marrow as well as for spleen cells the total duration of the cell cycle did not differ from the cell cycle times of normal erythroblasts. From the difference between the actual doubling time and the potential doubling time (estimated on the basis of the cell cycle time) it can be calculated that considerable cell loss must occur. This cell loss is only to a minor extent due to the release of blasts into the peripheral blood. Probably cell death and extrusion of nuclei during erythroid differentiation are the main factors involved.

Modulation of fibroblast hemopoietic regulatory activities by infection with cloned Rauscher helper leukemia virus.

We tested the ability of cellularly cloned Rauscher helper leukemia virus to modulate the release of hemopoietic regulatory activities by skin fibroblasts in culture. The results demonstrate that release of colony stimulating activity for granulocyte/macrophage progenitors by fibroblasts derived from BALB/c, NIH/RIV, 129/J, and DBA/2 mice was increased by virus infection. In contrast, virus infection severely impaired the ability of fibroblasts to support in-vitro granulopoiesis.

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.

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.

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.

Influence of the microenvironment on invasiveness of human bladder carcinoma cell lines.

To investigate the importance of the microenvironment in bladder cancer invasion, a panel of six bladder carcinoma cell lines (SD, RT112, JON, 1207, T24, and J82) was tested in both in vitro and in vivo invasion assays. Furthermore, invasiveness was correlated with the expression of components of the E-cadherin-catenin complex. The E-cadherin-negative cell lines, T24 and J82, displayed a high in vitro invasive capacity, whereas the E-cadherin-positive cell lines, SD and JON, completely lacked in vitro invasive capacity. In contrast, in vivo invasion was noted for all cell lines, with the exception of cell line JON. Most notably, SD formed highly invasive tumors in vivo. The in vivo invasiveness of the E-cadherin-positive bladder carcinoma cell lines was associated with a heterogeneous expression of the E-cadherin-catenin complex. The discrepancy between in vitro and in vivo invasive behavior implies that, in vivo, the microenvironment plays an important role in the establishment of the invasive phenotype. In addition, it was found that orthotopic xenografting of 1207 and T24 bladder carcinoma cells resulted in site-specific tumor take and an enhanced tumor outgrowth and invasiveness, respectively, compared with heterotopic (i.e., subcutaneous) inoculation. We conclude that the site-specific growth and invasion of the bladder carcinoma cell lines in vivo and the observed assay specific invasion (in vitro vs in vivo) points to an effect of the local (bladder) microenvironment on tumor cell behavior.

Establishment of cell lines from adenocarcinomas of the esophagus and gastric cardia growing in vivo and in vitro.

The aim of this study was to establish cell lines of adenocarcinomas of the gastro-esophageal junction(GEJ), which grow in vivo and in vitro. Primary esophageal and gastric cardia adenocarcinomas and corresponding lymph node metastases were xenografted subcutaneously to immunodeficient nude mice. In addition, tumor tissue was also used for in vitro culture. Xenografting of 70 primary adenocarcinomas and 17 metastases resulted in the initial growth of 22 and 6 tumors, respectively (total 32%). Upon retransplantation, six long-term xenografts [esophageal adenocarcinoma (OAC)P33X, OACP47X, OACP56X, OACP58X, OACP67X, OACP76X] from primary tumors and three (OACM2.1X, OACM30X, OACM53X) from metastases were obtained. In vitro culture attempts of 34 primary tumors and nine metastases resulted in the establishment of three (7%) permanent in vitro growing cell lines. From one patient, a cell line from the primary tumor (OACP4 C) and from a lymph node metastasis (OACM4.1 C) was established. The third cell line (OACM5.1 C) was also derived from a lymph node metastasis. The in vivo and in vitro cell lines were characterized using immunocytochemistry and microsatellite analysis to verify their epithelial and human tumor origin, respectively.

Evaluation of oncogene amplification in intact and truncated cell nuclei of gastro-esophageal cancer cell lines by DNA in situ hybridisation.

Adenocarcinoma arising around the gastro-esophageal junction (GEJ) is a highly malignant form of cancer. Its incidence is rising sharply. The study of oncogenes in these carcinomas may give information concerning treatment and prognosis. In the present study, the fluorescence in situ hybridisation (FISH) technique was optimised for genetic characterisation of oncogenes in archival cancer specimens. Three cell lines derived from GEJ adenocarcinomas were investigated, i.e. JROECL 19, JROECL 33 and OACM5.1C, both in fresh and paraffin-embedded preparations. Furthermore, paraffin-embedded material of three xenografts was studied, i.e. JROECL 19, JROECL 33, and OACM4.1X. We focussed on the oncogenes MYC and HER2/neu, since they are frequently involved in intestinal cancers. Firstly, our results indicate that it is feasible to detect oncogene-specific probes with the FISH technique in formalin-fixed, paraffin-embedded material. Secondly, it appeared that the optimal section thickness for analysis was 2 microm. This thickness resulted in minimal nuclear overlap, which facilitates counting of FISH spots. Due to the truncation phenomenon, however, the sensitivity of the technique is less than FISH on intact nuclei. Importantly, (high level) oncogene amplifications were easily recognised in 2 microm thick sections. Finally, counting of the individual copy number of the MYC and HER2/neu oncogenes was feasible enabling an arbitrary assessment of low- and high-level amplification. In conclusion, FISH is an accurate technique for detecting amplification of oncogenes in paraffin-embedded patient material.

[Therapeutic cloning: far from application at this stage]. / Therapeutisch kloneren: nog verre van toepasbaar.

Therapeutic cloning has become possible since the discovery that nuclei from somatic cells of adult animal tissue can successfully be used for cloning and the fact that human embryonic stem cell lines have been established from preimplantation embryos. When nuclei from healthy tissue of a patient are transplanted into enucleated oocytes, these oocytes can be artificially activated so that embryos develop from which embryonic stem cells of the donor can be derived. These embryonic stem cells can be cultured as permanent lines in unlimited numbers and remain pluripotent, i.e. they can be induced to differentiate into the required cell type by adding one or more specific factors. These cells can then be transplanted back into the patient suffering from either a lack or dysfunction of these cells. This approach prevents the rejection of the transplanted cells by the patient's immunological system. As this type of cloning has a very low efficiency, a large number of unfertilized donor oocytes is required. It is questionable whether enough donors are or will be available for this purpose. The cultured cells must satisfy certain conditions before they can be used for transplantation. They must be checked for chromosomal abnormalities, and a complete differentiation of the embryonic stem cells into the cells types needed by the patient is necessary as after the transplantation, undifferentiated stem cells will form teratomas. Furthermore, it is difficult to ensure that the cells end up in the right place and to ensure that they fully integrate into the existing tissue to form functional connections. Due to this array of technical problems the question remains as to whether therapeutic cloning will become feasible in the near future.

The developmental potencies of the regeneration blastema of the axolotl limb.

1. The developmental potencies of limb regeneration blastemas of the axolotl (Ambystoma mexicanum) were tested by transplanting them to the flank or to the orbit under various experimental conditions. 2. Early upper-arm blastemas transplanted singly to the damaged flank musculature or to the orbit can form forearm skeletal elements in addition to hand cartilages (digital and carpal elements). 3. Fusion of the mesenchyme of several early upper-arm blastemas into one transplant leads to the formation of a single regenerate. In this case an upper-arm element may differentiate in addition to the more distal limb regions. 4. In both transplantation sites single hand plates of paddle-shaped upper-arm blastemas form exclusively hand structures. Combination of mesenchyme of several hand plates, however, can result in the differentiation of forearm elements in addition to hand structures. In one case even an upper-arm element was formed. 5. The single transplantation of carpal blastemas of various stages leads to the development of hand structures only. Combination of the mesenchyme of several carpal blastemas on the flank shows no improvement of regional differentiation. When, however, the same combination experiment is performed in the orbit, the carpal blastemas of the oldest stage used are able fo form forearm elements as well. 6. The first conclusion that can be drawn from these results is that the early upperarm blastema possesses the information for the development of all the structures lost by amputation. Morphogenetic stump influences are not necessary for the formation of these structures. 7. The second conclusion is that the limb pattern in the transplants is established autonomously, because mesenchyme of several blastemas combined in random orientation gives rise to a single, normally oriented regenerate. 8. The third conclusion is that mesenchyme which is already differentiating into hand structures at the time of transplantation (hand plate of paddle-shaped upper-arm blastema, and oldest carpal blastema) upon dedifferentiation can be induced to form more proximal structures as well. Therefore this mesenchyme still possesses the information for the development of structures which it would never have formedin situ. 9. In the transplants that do not regress completely digital elements are always formed. According as regression is less extensive, or the mass of transplanted mesenchyme is enlarged experimentally, carpal elements are realized next, then forearm elements, and finally also the upper-arm element. This suggests that in the normal blastema distal differentiation tendencies appear first - under the influence of the epidermis - while successively more proximal differentiation tendencies arise as the blastema increases in mass. The disto-proximal succession of differentiation tendencies would continue until definitive differentiation begins in the most proximal mesenchyme, whence it proceeds in a proximo-distal direction. 10. Since transplanted mesenchyme can form more structures than are lost by amputation if its mass is increased, it is likely that during normal regeneration the level of amputation determines what will be formed by regulating the mass of the blastema, while inhibitory stump influences may prevent the development of supernumerary structures from the blastema.

Epithelial regeneration of transposed intestine after high doses of X-irradiation.

The regeneration capacities of normal and transposed small bowel epithelium were compared in rats after applying high doses of X-irradiation. It has been shown that the potency of the mucosa to regenerate is much higher than assumed and that the mucosa can regenerate after single doses varying from 2000-5000 R. Even in the villus epithelium and in flat epithelium covering infiltrates of the lamina propria cells survive, which are still able to resume proliferative activity several days after irradiation.

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

The 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, auxiliary role during antibody-mediated, tumor destruction in our model.

A Rauscher-virus-induced T-lymphocyte cell line. Induction of differentiation under influence of dimethylsulfoxide and phorbolesters.

DBA/2 mice which are neonatally infected with Rauscher helper virus (R-MuLV) develop predominantly lymphatic leukemias. From one of these lymphatic leukemias we established a permanent cell line which we named RLD (Rauscher Lymphoid DBA/2). Phenotyping of this cell line with a panel of monoclonal antibodies directed to cell-surface determinants shows that RLD cells have T-cell characteristics: they bind monoclonal antibodies directed to the antigens Thy-1, T-200 and Lyt-1; they do not react with anti-Lyt-2 antibodies, nor do they react with antibodies directed to determinants on B cells or myelomonocytic cells. RLD cells show a high activity of the nuclear enzyme terminal deoxyribonucleotidyl transferase (TdT). RLD cells are able to differentiate after in vitro stimulation with 1% DMSO or with 30 nM tetradecanoylphorbol-1.3-acetate (TPA). This differentiation process is reflected by (1) changes in the 2D gel electrophoresis pattern of metabolically labelled proteins, (2) a decrease in TdT activity and (3) changes in the expression of cell-surface markers. Flow cytometric analysis of stimulated RLD cells shows a strong increase in the Lyt-1 expression. Together these data indicate that RLD cells are immature T lymphocytes which upon appropriate stimulation differentiate along the line of T helper cells.

Clonal growth of colorectal-carcinoma cell lines transplanted to nude mice.

It is generally assumed that tumor progression is a microevolutionary process in which increasingly aggressive clones, generated through genetic instability, emerge in an initially monoclonal lesion. The present study was undertaken to determine how rapidly a dominant clone will emerge from an initial polyclonal situation, and whether dominance of these clones is a prerequisite for the onset of metastasis. To this end, colon-carcinoma cells were infected in culture with an amphotropic retroviral vector containing the neomycin-phosphotransferase gene, which makes cells resistant to neomycin. A heterogeneous population of neomycin-resistant cells carrying random retroviral integrations was xenografted to the subcutis and to the cecum of nude mice. The xenografts obtained, as well as the available metastases, were analyzed as to viral integrations by Southern blotting. The results show that, (i) clonal selection already takes place during growth of the primary tumor; (ii) dominant clones also generate metastases. The retroviral integration pattern of metastases turned out to be identical to that found in the primary xenografts. This pattern remained unchanged in tumors obtained after serial transplantations of cells cultured from metastases.