[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.

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.

Genetic alterations involving exon 3 of the beta-catenin gene do not play a role in adenocarcinomas of the esophagus.

Beta-catenin has been identified as an oncogene. Phosphorylation of sites encoded by exon 3 of the beta-catenin gene facilitates degradation of this protein by the adenomatous polyposis coli (apc) gene product. Mutations in these sites or inactivation of apc lead to stabilization of beta-catenin, which then translocates to the nucleus where it modulates the transcription of genes involved in tumor formation. To explore the role of beta-catenin mutations in adenocarcinomas of the esophagus, we screened for genetic alterations in exon 3 in 69 tumor samples. We detected no mutations in exon 3 by PCR-SSCP analysis nor did we find large interstitial deletions involving exon 3. beta-catenin immunostaining on 54 tumors showed focal nuclear staining in 7 tumors and homogeneous nuclear staining in 3 tumors; in the latter; no mutations in the mutation cluster region of apc were detected. These results show that genetic alterations of exon 3 of the beta-catenin gene do not occur and therefore do not contribute to the pathogenesis of esophageal adenocarcinomas. The abnormal cytoplasmic and nuclear localization of beta-catenin indicates that other mechanisms leading to elevated free beta-catenin in these cancers must be involved.

Phenotyping of Evi1, Evi11/Cb2, and Evi12 transformed leukemias isolated from a novel panel of cas-Br-M murine leukemia virus-infected mice.

Cas-Br-M murine leukemia virus (MuLV) is a slow-transforming retrovirus that potently induces leukemias in mice and therefore is well suited for retroviral insertional mutagenesis. We used Cas-Br-M MuLV in NIH/Swiss mice to establish a new panel of mainly myeloid leukemias. All tumors found in leukemic animals were classified by gross pathology, morphology, and immunophenotype, as well as the incidence of known common virus integration sites (VISs) in MuLV-induced myeloid malignancies (i.e., Evi1, Evi11/Cb2, Evi12, Fli1, and c-Myb). Interestingly, male mice were more susceptible than females to the induction of leukemia by Cas-Br-M MuLV. Seventy-four of the Cas-Br-M MuLV-inoculated mice developed a severe splenomegaly, sometimes in association with a thymoma. Although most of the immunophenotyped Cas-Br-M MuLV tumors were of myeloid origin (58%), numerous T-cell leukemias (21%) and mixed myeloid/T-cell leukemias (21%) were found. The myeloid leukemias and myeloid compartment of the mixed leukemias were further characterized by immunophenotyping with stem cell-, myeloid-, and erythroid-specific antibodies. The known Cas-Br-M MuLV common VISs (Evi1, Evi11/Cb2, and Evi12) were demonstrated in 19%, 12%, and 20% of the cases, respectively, whereas no Fli1 and c-Myb rearrangements were found. Integrations into Evi1 were restricted to myeloid leukemias, whereas those in Evi11/Cb2 and Evi12 were identified in myeloid as well as T-lymphoid leukemias. This panel of well characterized Cas-Br-M MuLV-induced hematopoietic tumors may be useful for the isolation and characterization of new proto-oncogenes involved in myeloid or T-cell leukemias.

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

Various 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.

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

Reduced 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.

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.

The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11.

A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.

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.

Enhancement and suppression of DTH reactivity to Rauscher murine leukaemia virus induced tumour cell lines.

Delayed-type hypersensitivity (DTH) to Rauscher murine leukaemia virus (R-MuLV) encoded or induced determinants was induced in mice by three syngeneic R-MuLV-induced tumour cell lines, i.e. a myeloid tumour, RMB-1, an erythroid tumour, RED-1, and a lymphoid tumour, RLD-1. DTH to subcutaneously (s.c.) administered RMB-1 cells appeared on day 4, with a maximum DTH response on day 6 or 7. The induction of DTH could be prevented by intravenous (i.v.) pre-immunisation with R-MuLV-induced tumour cells several days before the s.c. immunisation. The three R-MuLV-induced tumour cell lines showed cross-reactivity in the DTH assay, whereas no cross-reactivity was found with syngeneic WEHI-3 cells. This indicates that the three R-MuLV-induced tumour cell lines share a virally encoded or induced antigenic determinant, which activates T-cells. When the RMB-1 cells used for immunisation had been cultured in medium supplemented with interferon-gamma (IFN-gamma), the subsequent DTH response was increased. This coincided with an increased expression of the R-MuLV-specific antigenic determinants on RMB-1 cells as demonstrated by Scatchard analysis. Furthermore, IFN-gamma increased the MHC class I antigen expression on RMB-1 cells, whereas the class II antigen expression remained undetectable.

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.

Molecular cloning of the Rauscher murine leukemia virus. Disease spectrum and integration pattern.

After transfection of NIH 3T3 cells with DNA from molecularly cloned Rauscher MuLV, virus was isolated which showed a disease spectrum comparable to that of R-MuLV cloned biologically by endpoint dilution. In both cases sites of proviral integration vary from 2-5 per leukemic tissue and occur apparently at random.

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.

Syngeneic monoclonal antibodies directed against Rauscher virus-induced myeloid leukemic cells: isolation and characterization.

Hybridomas producing syngeneic monoclonal antibodies (MAbs) were prepared by fusion of spleen cells of BALB/c mice, which were immunized with sublethal doses of RMB-I cells. This cell line originates from a Rauscher virus (R-MuLV)-induced myeloid leukemia and forms tumors when re-inoculated into mice. MAbs were characterized as regards their reactivity against virally and non-virally induced cell lines. Two selected MAbs, IC5F5 and 4D2B4, were analyzed further. Their binding to subcellular structures was determined, and so were the properties of the antigens to which they are directed. MAb IC5F5 is of the IgG2A and 4D2B4 of the IgG2b subclass. Both bind to R-MuLV-infected or -transformed cell lines and are not mutually competitive. The antibodies do not react with other murine and human myeloid leukemic cells. As shown by immuno-electron microscopy, these MAbs have affinity to the cell membrane of non-virus producing RMB-I cells. When lysates of purified virus were analyzed, the MAbs were found to be directed to the gag precursor protein Pr65, and one of them (IC5F5) also to be directed to the core protein p12. In RMB-I cells, binding occurs to a 50-kDa glycoprotein and 2 proteins of 26 and 29 kDa. Since RMB-I cells do not produce virus, but express aberrant viral proteins, these MAbs are tumor-specific and useful for immunotherapy.

Use of syngeneic monoclonal antibodies in the therapy of disseminated myeloid leukemic cells.

A syngeneic monoclonal antibody (MAb) (IC5F5) was successfully used in the immunotherapy of Rauscher-virus-induced myeloid leukemic RMB-I cells. It is directed to a virus-encoded, but aberrantly processed protein, which is expressed on the cell membrane. When applied in vivo, it binds only to RMB-I tumor cells. BALB/c mice were inoculated i.p. or i.v. with 10(7) RMB-I cells and died within 2-3 weeks due to increasing tumor load. Mice inoculated i.p. were completely cured by daily injections of ascites containing IC5F5. Disseminated tumor cells in liver and hemopoietic organs were observed after i.v. inoculation. Daily treatment with MAbs resulted in survival beyond 90 days. No antigenic modulation was observed when tumor tissue was analyzed 2-10 days after treatment. Treatment was successful even when therapy was postponed until day 5 following inoculation of tumor cells. When the number of ascites injections was reduced, survival was identical to that observed among repeatedly treated mice. Ten- and 100-fold dilution of ascites fluid diminished the number of survivors, but still resulted in a median survival time of 38 and 20 days, respectively, as compared to 14 days for untreated mice.

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies.

The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes 125I and 131I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with 131I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with 125I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.

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.