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Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This oncogene is generated by the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus (ABL) genes and encodes a novel fusion gene translating into a protein with constitutive tyrosine kinase activity. The discovery and introduction of tyrosine kinase inhibitors (TKIs) irreversibly changed the landscape of CML treatment, leading to dramatic improvement in long-term survival rates. The majority of patients with CML in the chronic phase have a life expectancy comparable with that of healthy age-matched individuals. Although an enormous therapeutic improvement has been accomplished, there are still some unresolved issues in the treatment of patients with CML. One of the most important problems is based on the fact that TKIs can efficiently target proliferating mature cells but do not eradicate leukemic stem cells, allowing persistence of the malignant clone. Owing to the resistance mechanisms arising during the course of the disease, treatment with most of the approved BCR-ABL1 TKIs may become ineffective in a proportion of patients. This article highlights the different molecular mechanisms of acquired resistance being developed during treatment with TKIs as well as the pharmacological strategies to overcome it. Moreover, it gives an overview of novel drugs and therapies that are aiming in overcoming drug resistance, loss of response, and kinase domain mutations.

INTRODUCTION Chronic myeloid leukemia (CML) is characterized by a pathognomonic chromosomal translocation, which leads to the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus 1 (ABL1) genes, generating an oncoprotein with deregulated tyrosine kinase activity. Areas covered: In the last two decades, BCR/ABL1 kinase has become the molecular target for tyrosine kinase inhibitors (TKIs), a class of drugs that impressively improved overall survival. Despite these results, a proportion of patients experiences resistance to TKIs and need to change treatment. Furthermore, TKIs are unable to eradicate leukemic stem cells, allowing the persistence of neoplastic clones. Therefore, there is still clinical need for new agents to overcome common resistance mechanisms to available drugs. This review explores the horizon of drugs actually under investigation for CML patients resistant to conventional treatment. Expert commentary: Radotinib is an ATP-competitive TKI that showed significant activity also in front-line setting and could find employment indications in CML. Asciminib, an allosteric ABL1 inhibitor, could demonstrate a higher capacity in overcoming common TKIs resistant mutations, including T315I, but clinical findings are needed. CML stem cell target will probably require new therapeutic strategies: combinations of several compounds acting with different mechanisms of action are actually under investigation.

To detect the expression of cancerous inhibitor of phosphatase 2A (CIP2A) in chronic myelocytic leukemia (CML) and investigate the mechanism underlying CIP2A knockdown-mediated cell proliferation and apoptosis as well as the interaction of CIP2A with breakpoint cluster region-Abelson leukemia virus (BCR-ABL). CIP2A mRNA and protein expression in chronic myelocytic leukemia-chronic (CML-CP) patients and healthy controls were determined by RT-PCR and Western blot. In vivo, c-Myc expression, PP2A activity, cell proliferation, and apoptosis of CML cells were detected with CIP2A depletion. In addition, the relationship among CIP2A, BCR-ABL, and tyrosine phosphatase SHP-1 was explored by depleting/overexpressing CIP2A or inhibiting BCR-ABL. The level of CIP2A mRNA was higher in CML-CP patients than healthy controls (56/74, 75.7 % vs. 1/35, 2.9 %, P < 0.001), and CIP2A protein was overexpressed in corresponding specimens. CIP2A knockdown by siRNA reduced the level of c-Myc protein and clonogenic formation, inhibited the activity of PP2A, K562 cell proliferation, and promoted cell apoptosis. Suppressing BCR-ABL by imatinib mesylate (IM) significantly decreased CIP2A expression. CIP2A knockdown decreased BCR-ABL but increased SHP-1 expression, and CIP2A overexpression had the reverse effect. CIP2A is overexpressed in CML-CP patients, and its expression may promote CML pathogenesis. CIP2A and BCR-ABL can regulate each other in a positive feedback loop. CIP2A may be a useful therapeutic target in CML-CP, particularly in patients with IM resistance. However, further studies are needed to validate the interaction between CIP2A and BCR-ABL using other tyrosine kinase inhibitors than IM.

Previously we have described the derivation of three distinct classes of leukemic cell clones from a single in vivo-passaged myelomonocytic leukemia, WEHI-274, that arose in a mouse infected with the Abelson leukemia virus/Moloney leukemia virus complex (K. B. Leslie and J. W. Schrader, Mol. Cell. Biol. 9:2414-2423, 1989). The three classes of cell clones were characterized by distinct patterns of growth in vitro, the production of cytokines, and the presence of cytokine gene rearrangements. However, all three classes of WEHI-274 clones bore a common rearrangement of the c-myb gene, suggesting that all were derived from the one ancestral cell and that at least three distinct and independent autostimulatory events were involved in the progression of a single myeloid leukemic disease. In this article, we demonstrate that the autocrine growth factor production by the WEHI-274 leukemic clones resulted from cytokine gene activations mediated by the insertion of an intracisternal A-type particle (IAP) sequence 5' to the interleukin-3 (IL-3) gene, in the case of the class I clone, or 5' to the gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), in the case of the class II clones. IAPs are defective murine retroviruses encoded by endogenous genetic elements which may undergo transpositions and act as endogenous mutagens. The functional IL-3 and GM-CSF mRNAs were generated by mechanisms in which the splice donor apparatus of the IAP sequence has been used in IAP gag-to-IL-3 or -GM-CSF splicing events.

The dose limitations imposed on cancer chemotherapeutic agents by their lack of selectivity can, in theory, be circumvented by a strategy entailing the prophylactic insertion into hosts of drug-sensitivity genes that are acquired or expressed in some but not all cells. This strategy predicts that neoplasms arising from drug-sensitive cells might be safely treatable with tumor-eradicative drug doses because the presence of a modicum of drug-insensitive stem cells will protect vital tissues from lethal depopulation. To test this prediction, lymphomas were induced with Abelson leukemia virus in mice bearing a herpes simplex virus thymidine kinase (HSV-TK) transgene selectively expressed in lymphoid cells. Of 12 transgenic mice treated with the HSV-TK-specific substrate ganciclovir (GCV), 11 exhibited complete tumor regressions; 5 of these mice remained tumor-free over observation periods that exceeded 100 days. Among the lymphomas that recurred, most appeared to represent mutant subpopulations that were GCV-insensitive because they had lost HSV-TK, implying that independent insertion of multiple HSV-TK gene copies might provide a means of preventing recurrences. The results of this study demonstrate that chemosensitivity genes can enhance the efficacy of treatment in hosts who subsequently develop a neoplasm. While the use of a germ-line gene insertion model precludes direct human application, the results also imply the merits of exploring an alternative version of the strategy in which somatic insertion of chemosensitivity genes in mosaic fashion is used prophylactically to enhance the prospect that a subsequent tumor will respond to therapy.

Although several murine macrophage (m4) cell lines from different sites have previously been ob- tamed by in vitro infection with theJ2 murin#{231} retrovirus, which carries the v-raf and v-myc oncogenes, it was not possible to immortalize thioglycolate-elicited peritoneal macrophages (Pm4s) by this in vitro procedure. A tech- nique utilizing in vivo injection of the J2 virus has been developed to overcome this problem. The J2 virus immor- talized Pm4s in a very efficient manner in vivo because no exogenous growth factors were required for the in vitro proliferation of these cells and numerous continuous cloned cell lines were readily established. In contrast, Pm4s obtained from uninfected mice or Pm4s infected in vitro with the J2 virus did not proliferate. The in vivo immortalized cells had many of the morphological and functional characteristics of m4s. Analysis of two of the clones, PMJ2-PC and PMJ2-R, demonstrated intracellular expression of the product of the v-raf gene, presence of m�-associated cell surface antigens, interleukin-6 secre- tion induced by lipopolysaccharide, and biological re- sponse modifier-induced cytotoxic activity against tumor cells. In addition, one of the clones, PMJ2-PC, constitu- tively expressed major histocompatibiity complex (MHC) class II antigens, and in the other clone, PMJ2-R, MHC class II antigen expression was induced by recombinant murine interferon-y. This method of utilizing the J2 virus in vivo represents a novel technique for obtaining hematopoietic cell lines from cells that are difficult to im- mortalize in vitro. J. Leukoc. Rio!. 53: 475-478; 1993. duced in vivo by Abelson leukemia virus (7), we explored the possibility of using the J2 virus to immortalize penitoneal macrophages (Pm4s) in vivo. In this report, we demonstrate that no immortalization of Pm4s resulted from in vitro infee- tion ofthese cells with theJ2 virus but, in contrast, injection of the J2 virus into the peritoneal cavity of mice resulted in the immortalization of cells that maintain many of the properties and functions of m4s. 2 virus was obtained from supernatants of exponentially growing cultures of AMJ2-C11 cells by a previously de- senibed technique (4). Three days after intrapenitoneal injec- tion with i ml of iO% Brewer's thioglycollate (Difeo, Detroit, MI), C57B1/6J mice (Jackson Laboratories, Bar Harbor, ME) were injected with 0.5 ml of the J2 viral suspension or medium alone (mock infected). Two days after infection the animals were sacrificed and Pm4s harvested as previously

Current studies have shown that all mammalian sarcoma-producing viruses, whether isolated from laboratory experiments of naturally occurring tumors, are deletion mutants of replicating mammalian type C viruses. Nonproducer cells transformed by any of these sarcoma viruses contain RNA homologous to mammalian leukemia viruses, even though the cells contain no known proteins currently coded for by the mammalian leukemia virus. This mammalian leukemia virus information (FT-) is a genetically stable part of the mammalian sarcoma viruses (FT+). Second, another component in the Kirsten and Harvey sarcoma viruses can be identified in addition to this leukemia virus information for the homologous leukemia virus; at least part of the additional information came from rat type C viruses from the animals in which the sarcoma viruses were isolated. This indicates that these two mammalian sarcoma viruses are recombinants between mouse leukemia virus and genetic information in rat cells and suggests that the process of formation of the sarcoma virus is analogous to transduction of information in bacteriophage. Third, the Kirsten sarcoma virus seems to have a third component in it separate from either the mouse leukemia virus or rat leukemia virus information. Fourth, and FT+ leukemia virus isolated from mice, the Abelson leukemia virus which causes as B-cell leukemia, is also defective and can be shown to have information homologous to Moloney leukemia virus. Fifth, in the feline sarcoma virus, feline leukemia information can be detected, but information for the other cat virus, RD-114, cannot be detected. Finally, mutants of Kirsten sarcoma virus temperature sensitive for the maintenance of transformation have been isolated, and out of ten such mutants, thus far no complementation has been observed.

While C-type RNA viruses are known to induce leukemias and lymphomas, oncogenic transformation of lymphoid cells by them in vitro has not been reported. In this study, splenocytes from female BALB/c mice were infected in vitro with Abelson virus, a C-type RNA virus that induces nonthymic lymphomas and plasmacytomas in mice. The cells were transplanted into recipients of different karyotype, either male BALB/c mice or hybrid BALB/c × AL/N (CALF1) mice, which bear the Rb(5.19) 1 Wh translocation. Transplants of eight of the resulting tumors (one plasmacytoma and seven lymphomas) contained cells of donor BALB/c karyotype, indicating that transformation of splenocytes occurred in vitro .

The isolation of two distinct classes of transformation-defective cell lines nonproductively infected with Abelson leukemia virus (AbLV) is described. One group was selected as spontaneous revertants of an AbLV-transformed mink cell line. Mutants of this group are shown to be defective both in transformation and in expression of gag gene proteins (p15 and p12) encoded by the amino terminal region of the AbLV genome. Superinfection of such cell clones by various helper viruses led to rescue of wild-type AbLV, arguing that the transformation defect in the morphologically reverted clones involves a defect in cellular genes influencing transformation rather than in the viral genome itself. Mutants of the second group were isolated by screening single cell clones, newly infected by AbLV pseudotype virus, for expression of p12 antigen in the absence of either transformation or virion production. Twenty such mutants were selected. All clones except one expressed high levels of p15 and p12 in the absence of detectable levels of other gag gene-coded structural proteins. One clone was also positive for envelope glycoprotein (gp70) apparently encoded by a replication-defective helper virus mutant. In the second group of mutant cell clones, attempts to isolate wild-type AbLV were unsuccessful. Moreover, transformation could be induced following superinfection with wild-type AbLV pseudotype virus but not with superinfection by helper virus alone. The possibility that these latter mutants may be viral rather than cellular in origin is discussed.

Spleen cell cultures stimulated to DNA synthesis by antigen or mitogen were infected with Abelson virus, a C-type RNA virus inducing nonthymic lymphomas in mice. After 3 days the cells were transferred to mice and caused 100 percent incidence of lymphomas in as few as 29 days. That a number of the tumors were of donor origin, as shown by female karyotypes in recipient male mice, indicated that cells infected by virus in vitro were transformed. The process depended upon both virus and stimulation of lymphocytes in culture. Lymphoid tumors did not develop in mice receiving cells from virus-infected cultures not exposed to antigen or mitogen.

An examination of Abelson murine leukemia virus (A-MuLV)-hematopoietic cell interaction in cultures of fetal tissues reveals that A-MuLV can stimulate the formation of two different types of colonies. One type of colony is white and composed of A-MuLV-transformed lymphoid cells that can develop into established cell lines. These cells are indistinguishable in morphology from typical adult-derived lymphoid transformants. The second type of colony is pink or red and composed of erythroid cells in various stages of differentiation. Although A-MuLV is required to induce the erythroid colonies, and at least some cells in all of these colonies are infected with the virus, no permanently growing cell lines have been established from the cells in these colonies. The frequency of the two types of colonies varies depending upon the tissue and the gestational age of the embryo. Erythroid colonies are found following infection of early and mid gestation tissues while lymphoid colonies are found following infection of mid and late gestation tissues. Mixing experiments indicate that the two types of colonies arise from distinct target cells. Because A-MuLV mutants that are defective for lymphoid cell transformation are also defective for erythroid colony induction, expression of a functional Abelson protein is probably required for colony induction. Thus A-MuLV is capable of stimulating the cells of two distinct hematopoietic lineages. In one case, infection leads to transformation, while in the second, it leads to growth and differentiation. Both types of interaction are mediated, at least in part, by the same A-MuLV gene product, a molecule previously considered to induce transformation in all stably infected cells.

The genomic RNA of Abelson leukemia virus (AbLV) has been purified and translated in Xenopus laevis oocytes. The primary AbLV-specific protein synthesized is a polyprotein corresponding in molecular weight and immunological properties to a previously described p15 and p12 containing 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells. In contrast, translation of woolly monkey sarcoma virus genomic RNA resulted in symthesis of a 55,000-molecular-weight polyprotein consisting of woolly helper virus p30, p15, and p12. These findings demonstrate the value of the X. laevis oocyte in vitro system for studies of translational products of replication-defective transforming viruses and establish the virus-coded nature of the nonstructural component of the 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells.

Summary A series of murine lymphosarcomas, including those induced by Abelson leukemia virus, were adapted to culture. Their cell growth was inhibited by B lymphocyte mitogens at concentrations which did not affect other hematopoietic cell lines: myelomas, T lymphomas, mastocytoma and erythroleukemia. The pattern of inhibition among different lymphosarcoma lines suggests that they represent tumors of B lymphocytes at different stages of differentiation.

A 16-year-old girl with an unusual presentation of lymphoblastic lymphoma is described. The tumor originated as a paraspinal mass accompanied by lytic bone lesions in the pelvic bones. Neither an anterior mediastinal mass nor lymphadenopathy were noted. This pattern of disease is strikingly reminiscent of lymphomas induced in mice by the Abelson leukemia virus. This patient's lymphoma cells, unlike the usual lymphoblastic lymphoma cells, did not bear classical T-lymphocyte surface markers. A permanent cell line was derived from her tumor. Sparse amounts of cytoplasmic mu-immunoglobulin heavy chains were present in most of these cells, suggesting that the tumor originated in pre-B-lymphocytes. The phenotype of the cultured tumor cells is similar to Abelson virus-transformed murine bone marrow cells. Tumor cells of this phenotype may originate in the bone marrow and spread subsequently in and around the involved bones. This pattern of disease is distinctly different from that of T-cell lymphoblastic lymphoma, which typically presents in the mediastinum and cervical lymph nodes, and involves the bone marrow only as a late manifestation of advanced disease.

The Abelson leukemia virus (AbLV) polyprotein P120 is compared to translational products representing the entire Moloney murine leukemia virus (MuLV) genome on the basis of [35S]methionine tryptic peptide composition. Three methionine-containing tryptic peptides present in Moloney Pr65gag are each shown to be present in both Pr75gag and in Pr180gag-pol. Of these, one peptide, corresponding to Moloney MuLV p12, but neither of two p30-specific peptides are present in AbLV P120. Among the 12 remaining methionine-containing peptides present in AbLV P120, many, if not all, are unique to AbLV P120 and not shared by either Moloney MuLV Pr180gag-pol or Pr82gag.

Abelson leukemia virus (A-MuLV) is an oncogenic murine retrovirus whose genome contains sequences homologous to those of a normal cellular gene, c-abl. It has been demonstrated to cause rapid transformation of several cell types, including pre-B lymphocytes, macrophages, and fibroblasts. More recently, A-MuLV has been reported to induce thymic tumors in a mouse strain (C57BL/Ka) previously thought to be resistant to disease induction. We showed that the masses occurring after intrathymic injection of the virus were composed of lymphocytes of a previously described immature T-cell phenotype. This phenotype has been defined here by flow cytometry of 10 primary tumor samples stained with antibodies to several thymocyte differentiation antigens. Hybridization of DNAs from these tumors with v-abl, immunoglobulin mu, and T-cell antigen receptor beta-chain probes confirmed the T-lymphoid, polyclonal nature of the primary tumor cells. The primary tumors were malignant, as clearly shown by reinjection into Thy-congenic host animals. Further, four Thy- in vitro cell lines derived from three tumors differed from the majority of primary tumor cells and were similar to previously described A-MuLV-transformed pre-B cells. The consistent T-lymphoid phenotype exhibited by primary A-MuLV thymomas may represent one stage of normal thymocyte differentiation.

Five different types of protein kinase acivities have been evaluated in cell lines from murine lymphomas induced by Abelson leukemia virus (A-MuLV), whose oncogene codes for a tyrosine protein kinase. Such activities were compared with those of normal cells and of cells transformed by Moloney leukamia virus (M-MuLV), lacking oncogene sequences in its genome. While cAMP-dependent protein kinase and casein kinase-1 do not undergo significant changes, casein kinase-2 rises in both A-MuLV and M-MuLV infected lymphocytes, becoming largely associated with the particulate fraction of transformed cells. Protein kinase-C on the other hand is unchanged in M-MuLV transformed cells but it undergoes a 2–3-fold increment in both soluble and particulate fractions of A-MuLV transformed lymphocytes, which also display high tyrosine protein kinase activity.

Abelson murine leukemia virus (A-MuLV) is a replication-defective retrovirus capable of rapidly inducing leukemia in mice as well as of transforming in vitro mouse bone marrow cells and fibroblasts [1, 2, 14]; the A-MuLV was derived by passage in vivo of the replication-competent Moloney leukemia virus (M-MuLV) and its genome codes for a single polypeptide which is a hybrid molecule containing a portion of the parental M-MuLV genome (gag gene) and a portion of the cellular gene termed abl [18]. This protein, which varies in size from 160 to 90 kilodaltons, depending on the specific A-MuLV strain, is associated mainly with the detergent-insoluble cell fraction and possesses a tyrosine kinase activity [4, 15, 19]. In this regard, the A-MuLV protein resembles the pp60src encoded by the Rous sarcoma virus (RSV) [10] and the protein kinases encoded by other retroviruses such as the feline sarcoma virus (FeSV) [3, 17] and the Fujinami sarcoma virus (FuSV) [9].

Friend murine leukemia virus (Fr-MuLV) is a replication-competent murine retrovirus that induces acute nonlymphocytic leukemias in NFS/n mice. Fr-MuLV disease is divided into two stages based on the ability of the leukemia cells to grow in culture and transplant into syngeneic mice. Hematopoietic cells taken from the early stage of disease after Fr-MuLV infection grow as immortal myeloid cell lines in the presence of WEHI-3 cell-conditioned medium (CM) or interleukin 3. These growth factor-dependent cell lines do not grow in culture in the absence of CM and do not form tumors in syngeneic animals. If these Fr-MuLV-infected cells are superinfected with Abelson murine leukemia virus (Ab-MuLV), they lose their dependence on WEHI-3 CM and proliferate in culture in the absence of exogenous growth factors. Concomitant with the loss of growth factor dependence in culture, the Ab-MuLV-infected cell lines become tumorigenic in syngeneic mice. This secondary level of transformation is Ab-MuLV specific. Fr-MuLV-immortalized myeloid cell lines superinfected with Harvey murine sarcoma virus (Ha-MuSV) or amphotropic virus remain dependent on WEHI-3 CM for growth in vitro and are not tumorigenic in vivo. Neither Ab-MuLV- nor Ha-MuSV-infected normal mouse myeloid cell cultures produce growth factor-independent or tumorigenic cell lines. We conclude that at least two genetic events are needed to convert a murine myeloid precursor into a tumorigenic cell line. The first event occurs in Fr-MuLV-infected mice, generating cells that are growth factor dependent but immortal in vitro. The second event, which can be accomplished by Ab-MuLV infection, converts these immortal myeloid precursors into growth factor-independent and tumorigenic cells.

MURINE leukaemia viruses (MLV) cause tumours of haemopoietic origin in vivo1,2, but although they replicate in vitro, they generally do not transform cells. MLV provides helper activity3 for the replication of defective murine sarcoma virus (MSV)4 which possess a transforming activity5. Abelson and Rabstein6 isolated an agent in association with Moloney leukaemia virus (MLV-M) which causes a rapidly progressive lymphoblastic leukaemia of bone-marrow-derived lymphocytes (B cells)7. In addition, this Abelson virus (MLV-A) in conjunction with MLV-M causes rapid appearance of immunoglobulin-producing plasmacytomas in BALB/c mice primed with oil8. We now report that MLV-A can be defective for virus replication and show that this agent directly transforms 3T3 cells in vitro. A quantitative transformation assay is described.

The onc gene abl is a member of the tyrosine kinase family of oncogenes and was first isolated in a murine retrovirus, Abelson leukemia virus (A-MuLV).(1) This virus has been of interest as one of a number of transforming viruses that encode tyrosine protein kinases. In addition, because of its ability to transform both fibroblasts and hematopoietic cells, A-MuLV has been a useful tool to probe growth and differentiation of hematopoietic cells and to examine questions of virus-target cell specificity. Since the isolation of A-MuLV, a second virus, HZ-FeSVII, containing a portion of the abl gene has been isolated from a spontaneous feline sarcoma.(2) In addition, analyses of c-onc genes revealed that the chromosomal translocation associated with human chronic myelogenous leukemia (CML)(3) alters the location and expression of the c-abl protooncogene.(4–6) Although in each of these settings, expression of the tyrosine kinase activity associated with the abl gene product(7–10) appears to play a key role in oncogenesis, the exact mechanism by which this is accomplished remains elusive. Because a general discussion of the origin and biology of A-MuLV has been presented recently,(11–13) this review will focus on recent developments concerning the transformation potential of v-abl genes and the expression of both the normal c-abl gene and the altered bcr/abl gene found in CML.

We have studied the nucleic acid sequences in nonproducer cells transformed by Moloney sarcoma virus or Abelson leukemia virus (two types of replication-defective, RNA-containing, viruses isolated by passage of Moloney leukemia virus in BALB/c mice). DNA probes from the Moloney leukemia in virus detect RNA in both Abelson virus-transformed nonproducer cells and Moloney sarcoma virus-transformed nonproducer cells. A sarcoma-specific cDNA, prepared from the Moloney sarcoma virus, has extensive homology to RNA found in heterologous nonproducer cells transformed by Moloney sarcoma virus, has little homology to RNA in cells producing Moloney leukemia virus, and no detectable homology to RNA in nonproducer cells transformed by the Abelson virus. By analogy to earlier data on avian and mammalian sarcoma viruses, these results suggest that the Moloney sarcoma virus arose by recombination between a portion of the Moloney leukemia virus genome and additional sarcoma-specific information, and indicate that the expression of this information in not essential for Abelson virus-mediated fibroblast transformation.

Hybrids between pseudodiploid Thy-1.1+ lymphomas and Thy 1.2- pseudodiploid Abelson-leukemia-virus-induced-(ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1+ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2+ or Thy 1.2-. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1+, Thy 1.2- hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1+ lymphomas and pseudotetraploid Thy 1.2- Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1+ revertants were isolated from one of the Thy-1- hybrids by cell sorting. - These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparently cis-active, regulatory mechanisms which determine the ability of the Thy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.

The chick chorioallantoic membrane (CAM) was used as an assay system to investigate the the invasive properties of viral transformed NIH/3Y3 cells. Scanning electron microscopy demonstrated that single Kirsten sarcoma virus (KiSV)-transformed cells passed between the epithelial cells of the CAM ectoderm within 6 hr of application, while viable NIH/3T3 cells did not penetrate the ectoderm within 24 hr. The transformed cells entered the mesoderm of the CAM and formed tumors of proliferating cells. The application of 5 X 10(5) KiSV-transformed cells resulted in the formation of donor cells resulted in the formation of the donor cell tumors within 5 days in 43% of the membranes. No tumors were formed when as many as 5 X 10(6) NIH/3T3 cells were applied to the membrane. NIH/3T3 cells transformed by the Abelson leukemia virus or the Moloney sarcoma virus also ivaded the CAM and formed tumors of proliferating cells within the mesoderm, while cells infected with the Moloney leukemia virus did not. NIH/3T3 cells inoculated onto the CAM 8 days after infection and transformation with KiSV formed tumors with a frequency similar to that of KiSV transformed cells that have been passaged in culture for many generations. Cells that formed invasive tumors within the mesoderm also attracted loops of host blood vessels.

Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing and phagocytosis of sheep erythrocyte targets, and secrete high levels of lysozyme. None of these properties was exhibited by the lymphocyte lines. Of the two macrophage cell lines tested, neither was capable of replacing the adherent cell population required for the induction of in vitro immune responses. An agent that activates normal macrophages, bacterial lipopolysaccharide, specifically inhibits the growth of the transformed macrophages in culture. Secretion of infectious Abelson leukemia virus by two of the macrophage lines, RAW 309Cr and WR 19M, provides conclusive evidence that the Abelson virus is capable of productively infecting the macrophage cell type. The other macrophage line, RAW 264, fails to secrete detectable virus particles and is negative in the XC plaque formation assay, as well as the fibroblast transformation assay for Abelson virus, but becomes positive for Abelson virus production after rescue by Moloney leukemia virus.