Abelson virus potentiates long-term growth of mature B lymphocytes.

Abelson murine leukemia virus (A-MuLV) infection of mouse bone marrow cells usually leads to transformation of pre-B cells. However, when the environment is modified by the continuous presence of lipopolysaccharide (LPS), two novel types of membrane immunoglobulin (mIg)-positive B cell lines are generated. Because the cells which give rise to these cell lines copurify with mIg-positive bone marrow cells, the cell lines arise as a result of A-MuLV interaction with a new type of in vitro target cell. The cell lines generated fall into two groups which differ in several phenotypic characteristics. Group 1 cells are more differentiated than the typical pre-B cell transformant in that they synthesize mIgM and appear to resemble virgin B cells. The group 1 cells do not secrete immunoglobulin and are independent of LPS for growth. In addition, these cell lines synthesize the Abelson P160 protein, contain integrated abl proviral DNA, and are highly tumorigenic in syngeneic animals. The group 2 cell lines differ markedly from both the group 1 cells and from typical, pre-B cell A-MuLV transformants. These cells are mIgG positive and secrete large amounts of immunoglobulin into the culture medium. The cell lines are comprised of both adherent and nonadherent cells and do not synthesize P160 or contain integrated v-abl sequences. The group 2 cells are nontumorigenic in syngeneic animals and require LPS for growth and viability. Both types of cells have remained in culture for over 2 years with no changes in their phenotypic characteristics. This A-MuLV infection system and the novel mIg-positive cell lines may serve as useful models for studying biochemical and molecular properties of mature B cells.     

Transformation of T-lymphoid cells by Abelson murine leukemia virus.

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.     

Clonal dominance and progression in Abelson murine leukemia virus lymphomagenesis.

We examined the clonality of tumors induced by an acutely transforming retrovirus which carries a single oncogene. Contrary to our expectation, tumors induced by the Abelson murine leukemia virus (A-MuLV) showed one to four major proviral integration events. To further investigate the process by which clonality was established, we analyzed the number of cells infected and transformed by A-MuLV at various times after in vivo infection. At the midpoint of tumor latency (14 days postinfection), we found that infection of total bone marrow cells by A-MuLV was efficient and polyclonal. However, only a minority of these infected cells were transformed as assayed in cell culture, and clonal dominance had already been established in this transformed cell population. Examination of the in vitro growth properties of transformed cells recovered from preleukemic and leukemic mice indicated that preleukemic cells had lower cloning efficiencies than primary tumor cells. Our results suggest that the rate-limiting step in this system of lymphomagenesis is the initial transformation of bone marrow target cells and that these cells undergo subsequent changes in cloning ability during the course of the disease that lead to an autonomous neoplastic state.     

Characterization of mouse cellular deoxyribonucleic acid homologous to Abelson murine leukemia virus-specific sequences.

The genome of Abelson murine leukemia virus (A-MuLV) consists of sequences derived from both BALB/c mouse deoxyribonucleic acid and the genome of Moloney murine leukemia virus. Using deoxyribonucleic acid linear intermediates as a source of retroviral deoxyribonucleic acid, we isolated a recombinant plasmid which contained 1.9 kilobases of the 3.5-kilobase mouse-derived sequences found in A-MuLV (A-MuLV-specific sequences). We used this clone, designated pSA-17, as a probe restriction enzyme and Southern blot analyses to examine the arrangement of homologous sequences in BALB/c deoxyribonucleic acid (endogenous Abelson sequences). The endogenous Abelson sequences within the mouse genome were interrupted by noncoding regions, suggesting that a rearrangement of the cell sequences was required to produce the sequence found in the virus. Endogenous Abelson sequences were arranged similarly in mice that were susceptible to A-MuLV tumors and in mice that were resistant to A-MuLV tumors. An examination of three BALB/c plasmacytomas and a BALB/c early B-cell tumor likewise revealed no alteration in the arrangement of the endogenous Abelson sequences. Homology to pSA-17 was also observed in deoxyribonucleic acids prepared from rat, hamster, chicken, and human cells. An isolate of A-MuLV which encoded a 160,000-dalton transforming protein (P160) contained 700 more base pairs of mouse sequences than the standard A-MuLV isolate, which encoded a 120,000-dalton transforming protein (P120).     

Abelson murine leukemia virus variants with increased oncogenic potential.

A number of strains of Abelson murine leukemia virus (A-MuLV) with various abilities to transform cells have been identified. Among these is the A-MuLV-P90 strain, a mutant derived from A-MuLV-P120 that encodes an A-MuLV protein missing sequences that are normally present at the extreme carboxy terminus of P120 (N. Rosenberg and O. N. Witte, J. Virol. 33:340-348, 1980). This virus transforms NIH 3T3 cells efficiently but does not transform a high frequency of lymphoid cells in vitro or in vivo. In this communication, we show that of the relatively few tumors induced by A-MuLV-P90 nearly all contained new variant viruses that stably expressed either larger or smaller A-MuLV proteins. Strains that expressed larger A-MuLV proteins behaved like A-MuLV-P120 in transformation assays, whereas those expressing smaller A-MuLV proteins induced a high frequency of tumors after a short latent period in vivo but failed to transform large numbers of lymphoid cells in vitro. Thus, these latter viruses separated the requirements for in vitro transformation of lymphoid cells from those for tumor induction. All of the variants differed from A-MuLV-P90 in the carboxy-terminal region of the A-MuLV protein, suggesting that sequences in this region play a key role in the ability of the virus to interact with hematopoietic cells in vivo and in vitro.     

Phenotypic variation in clonal Abelson virus lymphoma cells

Two clonal A-MuLV lymphoma cell lines have the capacity to generate phenotypic variants when grown in vivo as ascites tumors. Variant lines differed from parental lymphoma cells in their expression of enzymatic or cell surface differentiation markers. Parental lines expressed the B220 and Lyb-2 glycoproteins characteristic of pre-B cells and bound B220-specific monoclonal antibodies such as 14.8. The parental cells expressed low levels of TdT activity but did not synthesize detectable mu-heavy chain, a cellular phenotype that may correspond to lymphoid progenitor cells. Three classes of phenotypic variants were recovered from the Thy-1- parental lines: 1) 14.8+, Lyt-1+, Thy-1- cells; 2) 14.8 +/-, Lyt-1+, Thy-1+ cells, and 3) 14.8-, Lyt-1+, Thy-1+ cells. Cell cloning experiments indicated that Thy-1+ variant cells can be recovered within 14 days of in vivo inoculation as a minor proportion (1/10(6] of the tumor cell population and subsequently become the predominant tumor cell population. These clonal tumor lines provide a model for the study of cellular and molecular alterations that occur during neoplastic differentiation and progression in the lymphoid system.     

Site-directed deletions of Abelson murine leukemia virus define 3'' sequences essential for transformation and lethality.

Abelson murine leukemia virus (A-MuLV) encodes a single protein with tyrosine kinase activity that can transform fibroblast cell lines in vitro and lymphoid target cells in vitro and in vivo. Expression of kinase-active A-MuLV protein can result in a deleterious effect on transformed fibroblast populations, leading to cell death or selection for nonlethal mutants of the virus. These mutants retain expression of the kinase activity but have lost large portions of the carboxy terminus of the Abelson protein. To more precisely map the sequences involved in this lethal effect, we have isolated a series of site-directed deletions from a DNA clone of the P160 wild-type strain of A-MuLV. In addition, a number of unexpected, spontaneous deletions occurring during transfection of NIH 3T3 cells were isolated. These deletions result in expression of carboxy-terminal truncated forms of the A-MuLV protein ranging from 130,000 to 84,000 in molecular weight. Analysis of the transforming and lethal activities of each mutant recovered in its RNA viral form shows that the transformation-essential and lethal-essential sequences do not overlap. These data and our previous work suggest that a function carried by the carboxy-terminal region of the A-MuLV protein acts in cis with the kinase-essential region to mediate the lethal effect.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

In vivo tyrosine phosphorylations of the abelson virus transforming protein are absent in its normal cellular homolog

The transforming gene product of the Abelson murine leukemia virus (A-MuLV) is a phosphoprotein encoded by combined viral and cellular sequences. Previous work has shown the existence of a serologically crossreactive normal cellular phosphoprotein called NCP150. We have utilized two-dimensional phosphopeptide mapping and phosphoamino acid analysis to compare the structures of NCP150 and wild-type and mutant forms of the A-MuLV protein labeled in vivo with ³²P-orthophosphate. This analysis demonstrated clear homology between NCP150 and wild-type A-MuLV protein, but a number of phosphorylation differences were seen. Among them, two specific tyrosine phosphorylations present in all transformation-competent Abelson proteins were not observed in NCP150. No other phophotyrosine-containing peptides were detected. In addition, transformation-defective mutants isolated from either the P120 or P160 wild-type strain lack phophotyrosine-containing peptides. Double-infection studies with such transformation-defective and transformation-competent AMuLV strains show that Abelson viral proteins may be substrates for their own tyrosine-specific kinase activity in vivo. These observations suggest that the phosphotyrosine kinase activity of the abl region may be controlled, and may function, differently in its viral and cellular forms.     

BCR-ABL and v-abl oncogenes induce distinct patterns of thymic lymphoma involving different lymphocyte subsets.

The human BCR-ABL oncogenes encoded by the Philadelphia chromosome (Ph) affect the pathogenesis of diverse types of leukemia and yet are rarely associated with T-lymphoid leukemia. To determine whether BCR-ABL kinases are inefficient in transforming T lymphocytes, BCR-ABL-expressing retroviruses were injected intrathymically into mice. Thymomas that expressed BCR-ABL kinase developed after a relatively long latent period. In most thymomas, deletion of 3' proviral sequences resulted in loss of tk-neo and occasionally caused expression of kinase-active carboxy-terminally truncated BCR-ABL oncoprotein. In contrast, deletion of 3' proviral sequences was not observed in thymomas induced with Abelson murine leukemia virus (A-MuLV). BCR-ABL viruses induced distinct patterns of disease and involved different thymocyte subsets than A-MuLV and Moloney murine leukemia virus (Mo-MuLV). While Mo-MuLV only induced Thy-1+ thymomas, v-abl- and BCR-ABL-induced thymomas often contained mixed populations of B220+ and Thy-1+ lymphocytes in the same tumor. In most v-abl and BCR-ABL tumors, Thy-1+ lymphoid cells expressed CD8 and a continuum of CD4 ranging from negative to positive. Conversely, Mo-MuLV thymomas contained distinct populations of CD4+ cells that were either CD8+ or CD8-. A-MuLV-transformed T-lymphoid cells did not express the CD3/T-cell receptor complex, while BCR-ABL tumors were CD3+. Thus, BCR-ABL viruses preferentially induce somewhat more differentiated T lymphocytes than are transformed by A-MuLV. Furthermore, rare B220+ lymphocytes may represent preferred v-abl and BCR-ABL transformation targets in the thymus.     

p53 deficiency increases transformation by v-Abl and rescues the ability of a C-terminally truncated v-Abl mutant to induce pre-B lymphoma in vivo

Abelson murine leukemia virus (A-MuLV) is an acute transforming retrovirus that preferentially transforms early B-lineage cells both in vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine kinase related to v-Src but containing an extended C-terminal domain. Many mutations affecting the C-terminal portion of the molecule block the pre-B-transforming activity of v-Abl without affecting the fibroblast-transforming ability. In this study we have determined the abilities of both wild-type and C-terminally truncated (p90) forms of v-Abl to transform cells from p53(-/-) mice. Lack of p53 increases the susceptibility of bone marrow cells to transformation by v-Abl by a factor of more than 7 but does not alter v-Abl's preference for B220(+) IgM(-) pre-B cells. p53-deficient mice have earlier tumor onset, more rapid tumor progression, and decreased survival time following A-MuLV infection, but all of the tumors are pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl transformation but play no role in conferring preferential transformation of pre-B cells. Surprisingly, the C-terminally truncated form of v-Abl (p90) transforms pre-B cells very efficiently in mice lacking p53, thus demonstrating that the C terminus of v-Abl does not determine preB tropism but is necessary to overcome p53-dependent inhibition of transformation.     

A rat model to study the role of STn antigen in colon cancer

Expression of the mucin-associated sialyl-Tn (STn) antigen has been associated with a decreased survival in patients with colorectal, gastric, and ovarian cancer. To better understand the role of STn antigen in tumor biology, we developed STn(+) (called LP) and STn(-) (called LN) clonal cell lines from a parental metastatic rat colon carcinoma cell line (LMCR). Both derivative cell lines exhibited identical proliferation rates in vitro. LP cells strongly expressed STn antigen both in vitro and in vivo, and were poorly tumorigenic when given to syngeneic rats. LN cells did not express STn antigen in vitro, but as in vivo tumors these cells rapidly acquired STn expression, readily formed tumors, and were highly lethal. When rats were given an otherwise lethal inoculum of i.p. LN cells, pre-immunization with synthetic STn antigen conjugated to keyhole limpet hemocyanin (STn-KLH) resulted in a 60% survival rate. When LN cells were injected subcutaneously in the presence of STn-KLH-sensitized lymphocytes, tumor growth was decreased. Distribution of STn antigen in normal organs of host rats is quite similar to that of humans. This model mimics human disease and should facilitate studies of mucin-associated antigens in tumor biology and the development of immunotherapeutic agents based on mucin-related antigens.     

N-terminal mutations activate the leukemogenic potential of the myristoylated form of c-abl.

The two major forms of the c-abl gene differ from their activated counterpart, the v-abl oncogene of the Abelson murine leukemia virus by the replacement of their N-terminal sequences with viral gag sequences. Overexpression of p150c-abl type IV in a retroviral vector similar to Abelson virus does not transform NIH 3T3 fibroblasts, even though it is expressed and myristoylated at levels comparable to pp160v-abl. Members of a nested set of deletion mutations of the N-terminus of c-abl type IV in this expression system will activate abl to transform murine fibroblasts. The smallest of these deletions, delta XB, efficiently transforms lymphoid cells in vitro and causes leukemia in vivo demonstrating that gag sequences are not necessary for abl-induced leukemogenesis. The delta XB mutation defines an N-terminal regulatory domain, which shares a surprising homology with chicken oncogene v-crk and phospholipase C-II. Although overexpression of the myristoylated form of c-abl does not transform cells, it nonetheless has a profound effect on cell growth.     

Addition of constitutive c-myc expression to Abelson murine leukemia virus changes the phenotype of the cells transformed by the virus from pre-B-cell lymphomas to plasmacytomas.

Abelson murine leukemia virus (A-MuLV), a retrovirus that expresses the v-abl oncogene, characteristically induces pre-B-cell lymphomas following in vivo infection of BALB/c mice or in vitro infection of suspensions of fetal liver or bone marrow cells. ABL-MYC, a retrovirus that expresses both v-abl and c-myc, induces solely plasmacytomas in BALB/c mice. To investigate how the addition of overexpression of c-myc to that of v-abl accomplishes this dramatic change in the phenotype of the cells transformed by these closely related retroviruses, we utilized helper-free A-MuLV (psi 2) and ABL-MYC (psi 2) in vitro to infect suspensions of cells from different lymphoid tissues and purified immature and purified mature B cells. As expected, A-MuLV(psi 2) induced only pre-B-cell lymphomas in vivo and in vitro when immature B cells were present. ABL-MYC(psi 2), on the other hand, produced only plasmacytomas, even when purified immature B lymphocytes were infected in vitro. Although the A-MuLV(psi 2)-induced pre-B-cell lymphomas express easily detectable levels of c-myc mRNA, maturation into more-mature forms of B lymphocytes is blocked. The constitutively overexpressed c-myc in the ABL-MYC retrovirus abrogates this block, permits maturation of infected immature B cells, and yields transformed plasma cells.     

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.     

Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.     

Osteoblasts are target cells for transformation in c-fos transgenic mice

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype.     

Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines

DNA modification accompanying immunoglobulin gene expression was examined in various Abelson murine leukemia virus (A-MuLV)-transformed cell lines, which were able to differentiate from the mu- to mu+ stage or to undergo an isotype switch during in vitro culture. The C mu genes were relatively demethylated in the A-MuLV-transformed cell lines examined irrespective of whether or not the C mu genes were expressed. Normal IgM-bearing B cells, as well as a T cell line, also showed a similar DNA methylation pattern and the C mu genes were relatively demethylated. In one of the mu+ clones, however, the expressed C mu gene was heavily methylated. The DNA methylation pattern did not change and remained hypermethylated before and after gamma 2b expression in the two cell lines which underwent class switch to gamma 2b during in vitro culture, suggesting that expression of the gamma 2b gene was not accompanied by demethylation of the C gamma 2b gene. Taken together, these results indicate that DNA demethylation within and around the CH gene may not be necessary for its expression.