BALB- and Harvey-murine sarcoma virus transformation of a novel lymphoid progenitor cell.

BALB- and Harvey-murine sarcoma viruses (MSV) comprise a family of retroviruses whose mouse- and rat-derived onc genes are closely related. These viruses induce sarcomas and erythroleukemias in susceptible animals. An in vitro colony assay that detects transformation of lymphoid cells by Abelson-murine leukemia virus was used to demonstrate that BALB- and Harvey-MSV transform a novel hematopoietic cell both in culture and in vivo. Bone marrow colony formation was sarcoma virus dependent, followed single-hit kinetics, and required the presence of mercaptoethanol in the agar medium. BALB- and Harvey-MSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The cells had a blast cell morphology and lacked detectable markers of mature cells within the myeloid or erythroid series. They also lacked detectable immunoglobulin mu chain or Thy-1 antigen, markers normally associated with committed cells of the B and T lymphoid lineages, respectively. However, the transformants contained very high levels of terminal deoxynucleotidyl transferase (TdT), an enzyme believed to be specific to early stages within the lymphoid differentiation pathway. This phenotype distinguishes these BALB- and Harvey-MSV transformants from any previously reported hematopoietic targets of transforming retroviruses, including the pre-B lymphoid cell transformed by Abelson-MuLV under identical assay conditions. These newly identified lymphoid progenitor cell transformants may provide an important means of studying early stages of lymphoid ontogeny and the possible role of TdT in lymphoid development.

Single-cell suspensions of NFS/N bone marrow were exposed to virus for 1 h at 37°C in the presence of 4 tLg/ml polybrene (Aldrich Chemical Co., Milwaukee, WI). After centrifugation, the cells were mixed with semisolid agarose medium containing RPMI 1640 medium supplemented with 20% fetal calf serum, 5 × 10 .5 M mercaptoethanol (ME), and 0.47% Sea Plaque agarose (FMC Corp., Homer City, PA) and plated at the appropriate concentration into 35mm petri dishes in a volume of 2 ml. Plates were cooled at 4°C to allow solidification of the agarose, and the cultures were incubated at 37°C in a 5% CO2 humidified incubator. 1 ml of agarose medium was overlaid at 5-d intervals. Visible colonies were scored at 12-18 d after infection.

Establishment of BALB-MSV-and Harvey-MSV-Transformed Hematopoietic Cell Lines.
Individual colonies were removed from the agarose plates and suspended in 1 ml of RPMI growth medium in 24-well microtiter plates (Costar, Data Packaging, Cambridge, MA). When the cell number in an individual well reached ~ 106 cells, the cells were transferred at a 1:2 split ratio. After an initial adaptation period of 1-6 wks, the suspension lines could be maintained by transferring at 2-3-d intervals (split ratio of 1:20-1:100). In some cases, bone marrow cell-conditioned medium or secondary adherent bone marrow cell feeder layers were used during an initial adaptation period needed to establish hematopoietic cell lines. Any clones that were exposed to normal bone marrow feeder layers were recloned in soft agar before further characterization.
For preparation of feeder layers or conditioned medium, NFS/N bone marrow cell suspen- 6 sions (10 nucleated cells/ml) were plated in 60-mm tissue culture plates in RPMI 1640 medium supplemented with 20% horse serum (Flow Laboratories, Rockville, MD) and 5 × 10 -5 ME. Nonadherent cells and medium were removed after 5 d, and medium containing 15% fetal calf serum was used thereafter. Conditioned medium was collected at 5-d intervals starting 10 d after initial cell plating. Secondary adherent bone marrow cultures were generated from 15- Lysozyme Analysis. Lysozyme was assayed by the lysoplate assay as described by Osserman and Lawlor (29). Egg white lysozyme was used as a standard. Levels of lysozyme were expressed in ~g/106 cells.
Detection of Thy-I Antigen. The presence of Thy-1 antigen was determined by an antibodydependent cytotoxicity assay. The complement-mediated lysis assay was performed essentially as described by Reddy  Radioimmunoassayfor Immunoglobulin I~ Chain. The amount of mouse immunogiobulin/~ chain present in cells was determined by competition radioimmunoassay as previously described (24). Biosynthetic Labeling and Immunoprecipitation. The procedures for metabolic labeling with 35Smethionine and immunoprecipitation were as previously described (11). Briefly, 10 v transformed hematopoietic or normal thymus cells were labeled with 200/~Ci 35S-methionine (New England Nuclear, Boston, MA) for a 3-h period. The cells were disrupted in 1 ml of staphylococcus A (Staph A) lysing buffer, clarified, and 0.2-ml samples were incubated with 10/~1 goat anti-calf TdT serum (P-L Biochemicals, Inc.) or normal goat serum overnight at 4°C. Antigen-antibody complexes were precipitated with protein A sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, NJ) by agitation for 15 min at 4°C, washed three times in Staph A buffer, resuspended in sample buffer, and boiled for 2 min. Reduced samples were analyzed on sodium dodecyl sulfate-polyacrylamide gels. Gels were then fixed, dried, and exposed to X-Omat R film (Eastman Kodak Co., Rochester, NY).

BALB-MSV Induces Growth of Hematopoietic Colonies in Infected Bone Marrow Cultures.
Several transforming retroviruses, including avian erythroblastosis (32), myelocytomatosis (33), myeloblastosis (34), and retieuloendotheliosis viruses (35), as well as mammalian Abelson (36) and myeloproliferative viruses (37) preferentially cause hematopoietic tumors. In some cases, tissue culture assays for transformation of hematopoietic target cells by such viruses have been developed. To investigate the effects of BALB-MSV on hematopoietic cells, we used a colony-forming assay devel- The colonies appeared golden and iridescent under the microscope, were round or oblong in shape, and possessed a compact morphology ( Fig. 1 A). Cells within the colonies were round in shape and nonadherent. By light microscopy, such cells exhibited an undifferentiated blast cell morphology ( Fig. 1 B). The majority of cells had a large eccentric nucleus with a scanty cytoplasmic rim lacking any granules. Occasionally, cytoplasmic vacuoles were observed. There were no apparent morphologic differences in cells from 20 different colonies analyzed, although some variations in cell size were noted.
The length of virus exposure required for optimal colony formation was found to be 1 h. Incubation for longer periods appeared to decrease cell viability and did not increase the number of colonies detected. Variation in the cell number plated into soft agar from 5 × 105 to 3 × 106 cells correlated directly with the number of colonies formed (data not shown). As shown in Fig. 2, there was a linear relationship between the number of colonies formed and the amount of virus inoculated. The one-hit titration pattern suggested that colony formation was dependent on infection with a single virus particle. Colony formation was considerably less efficient than was focus formation in fibroblasts by the same BALB-MSV stock (Fig. 2). This may be due to a limited number of susceptible target cells for infection and/or transformation within the bone marrow cell population.
BALB-MSV, like other replication defective transforming retroviruses, can only be propagated in the presence of a type C helper virus. As shown in Table 1  activity must be due to BALB-MSV. We also investigated the ability of Harvey-MSV and Abelson-MuLV to induce hematopoietic colonies. Harvey-MSV is a rat-derived sarcoma virus (39, 40) whose onc gene is closely related to that of BALB-MSV (8).
Like BALB-MSV, Harvey-MSV was found to induce the formation of hematopoietic colonies (Table I). Under the same assay conditions, Abelson-MuLV induced colonies with indistinguishable morphology and at a comparable efficiency.

In Vivo Infection with BALB-and Harvey-MSV Leads to Induction of Similar Hematopoietic
Colonies. In view of our findings that BALB-and Harvey-MSV induced hematopoietic cell colonies in tissue culture, we investigated whether such colonies could be detected in virus inoculated animals. Moribund mice, killed 2-3 weeks after infection as newborns, demonstrated grossly enlarged spleens containing many pale, poorly defined foci. We also observed gross enlargement of the thymic region, which has not been previously reported. This pathology was not observed in helper virus-infected or -uninfected animals. When bone marrow cells from BALB-or Harvey-MSV infected animals were plated into soft agar, we observed the formation of compact colonies as early as 3-4 d after plating. As shown in Table II, the frequency of colony-forming cells ranged from 3.9 × 101 to 1.6 × 102 colonies per 106 nucleated bone marrow cells plated. The frequency of colony-forming cells in the bone marrow cells of Abelson-MuLV infected mice sacrificed at the same time was 5-15-fold greater.

Establishment and Characterization of Transformed Lines from BALB-and Harvey-MSVinduced
Colonies. The ability of a transforming virus to induce hematopoietic colony formation in tissue culture might reflect its direct transforming action or result from some indirect effect of virus infection such as the production of soluble factors that induce normal cellular proliferation. To differentiate between these two alternatives, it is necessary to determine whether such colonies are capable of continued, independent proliferation. Moreover, if cell lines can be established, it is possible to characterize their phenotype with respect to malignant potential and stage of differentiation. Thus, efforts were undertaken to develop clonal cell lines from colonies induced by BALB-and Harvey-MSV.
Individual colonies were transferred to liquid medium in microtiter wells and grown up to mass culture. The success rate for establishment of continuous cell lines could be improved with the initial use of conditioned medium or feeder layers. After an adaptation period of 1-6 wk, the transformed lines could be propagated independently. Any cell line established in the presence of a feeder layer was recloned in soft agar before its further characterization.
The established lines grew to saturation densities of-2 × 10 6 cells/ml and possessed generation times of 10-12 h. Individual cell lines formed colonies in soft agar at efficiencies ranging from 10-3 to 10 -1 (data not shown). Some clonal lines have been in continuous culture for >8 mo. Each cell line tested was found to release transforming virus. Under the assay conditions used, many of the colonies selected were likely to have been infected with both sarcoma and helper virus. Nonetheless, we would have expected to obtain some nonproductively transformed lines from colonies that arose at low multiplicities of infection. Thus, there may be some selective advantage for productively infected cells to develop into established lines.
To analyze the malignant potential of the cell lines, we inoculated NFS/N weanling mice subcutaneously with 5 × 106 cells from representative BALB-and Harvey-MSV transformed lines. Each cell line tested was oncogenic, forming very large tumors within 1-2 wk. Histopathologic analysis revealed the tumors to be comprised of undifferentiated hematopoietic blast cells. In contrast, subcutaneous inoculation of weanlings with 5 × 105 FFU of virus alone yielded either no detectable tumors or very small sarcomas, rather than hematopoietic tumors. To confirm the donor origin of the hematopoietic tumors, cells from BALB-and Harvey-MSV-transformed hematopoietic clones derived from male mice were injected subcutaneously into 5-wk-old female mice. The tumors that formed displayed a male karyotype. Moreover, a metacentric chromosomal marker identified in one of the donor clones was shown to be present in the tumor cells derived from the recipient mouse. All of these results established that the hematopoietic lines derived from BALB-and Harvey-MSV colonies were leukemogenic.

Phenotype of BALB-and Harvey-MSV Hematopoietic Transformants. To define the phenotype of BALB-and Harvey-MSV-transformed hematopoietic cells, we analyzed clonal lines for markers that help to distinguish cells of various hematopoietic lineages.
There was no evidence of hemoglobin synthesis as detected by benzidine staining (25), nor could the cells be induced to synthesize hemoglobin after exposure to DMSO or erythropoietin (Table III). The cells also lacked detectable nonspecific esterase or the ability to phagocytize latex beads, markers of cells within the myeloid series (Table  III).
To further define their stage of hematopoietic cell differentiation, we analyzed a number of individual clonal transformants for markers found associated with lymphoid cells. ME has been shown to enhance normal lymphoid cell survival and growth in tissue culture (41,42), as well as the growth capacity of certain lymphoblastic and myeloblastic tumor lines (43). ME has also been shown to be critical for hematopoietic cell colony formation by Abelson-MuLV and growth of Abelson-MuLV transformed lymphoid cells in culture (44,38). The growth of BALB-and Harvey-MSV transformants was generally found to be dependent on the presence of ME (Table III).
As shown in Table III Pre-B lymphoid cells, which synthesize heavy/x chain in the absence of detectable light chain (46), appear to be the preferential target for Abelson-MuLV transformation (47). When BALB-and Harvey-MSV transformants were analyzed for expression of mouse/~ chain, none contained detectable levels as determined by competition radioimmunoassay. In contrast, 5 out of 12 Abelson-MuLV in vitro transformed hematopoietic lines established in our laboratory produced readily detectable amounts procedures. Amounts of lysozyme were determined per 106 cells added, using purified egg white lysozyme as a standard. The sensitivity of detection was 0.5 /tg. Histochemical staining for nonspecific esterase was performed as described (26). Phagocytosis of latex beads was determined as described in Materials and Methods. Cultures were incubated for 1 h at an input of 500 beads/cell. Cells that internalized -> 10 beads were scored as positive. § Exponentially growing clonal lines were transferred at a l:20 split ratio into separate plates. ME was omitted from the growth medium in one plate. Cultures were passaged every 3rd d and maintained for several weeks. Viability was monitored every 3rd d by trypan blue staining. Cultures were discarded when viability decreased to <5%. The presence of Thy-1 antigen was determined by complement-mediated lysis as described in Materials and Methods. Complement or anti-Thy-1 antibodies alone did not exhibit any cytotoxicity (<2% cell death). A Moloney-MuLV lymphoma cell line exhibited >99% lysis, whereas a Rauscher-MuLV pre-B lymphoma cell line demonstrated <2% cell death. The presence of Fc receptors was determined by the rossette assay as described in Materials and Methods. A cell line was considered positive if 5-10% rosette formation was reproducibly detected. SRBC alone or a Moloney-MuLV T lymphoma cell line did not yield any rosettes whereas a Rauscher-MuLV pre-B lymphoma cell line demonstrated 65% rosette formation. Cytoplasmic/~ chain synthesis was analyzed by radioimmunoassay as described (24). The sensitivity of/~ chain detection was -0.3 ng/mg of soluble cell protein. Cell lines were considered positive for TdT if they contained >0.1 U/10S/h as determined by enzymatic assay.
of/z chain (data not shown). The lack of # chain expression among the BALB-and Harvey-MSV-transformed hematopoietic cell lines suggested that these cells might be at an earlier stage of differentiation than pre-B lymphoid cells transformed by TdT activity is a characteristic of immature lymphoid cells of both T and B cell lineage (48)(49)(50). As shown in Table IV  several independent transformants analyzed. These levels were as high or higher than the TdT activity detected in normal thymus cells from 2-3-wk-old NFS/N mice (Table IV) (51). In contrast, Abelson-MuLV-transformed cell lines of both #+ and /z-phenotypes contained very low level of TdT, ranging from 0.02 to 0.01 U/10 s cell/ h, and TdT activity was undetectable in a Friend erythroblast cell line, clone 745 (Table IV). To examine the specificity of the enzymatic assay, cell extracts were incubated with antiserum to TdT before performing the enzymatic assay. This decreased incorporation of dGTP into acid-insoluble polymer by two-to threefold. A similar decrease was observed when purified TdT was tested under the same conditions (data not shown).
Murine TdT is known to have a molecular weight of 60,000 (52). As an independent means of identifying and quantitating the TdT activity in BALB-and Harvey-MSV transformants, we performed radioimmunoprecipitation analysis. When ~S-methionine-labeled cell extracts were exposed to anti-TdT serum, a 60,000-mol wt protein was precipitated from a BALB-MSV transformant (Fig. 3). Similar results were observed with extracts from normal thymus. In contrast, TdT was not detectable in an Abelson-MuLV transformed pre-B cell line tested under the same conditions. These findings help to establish the identity of our BALB-and Harvey-MSVhematopoietic transformants as early cells within the lymphoid differentiation pathway as well as distinguishing them from any previously reported targets of transforming retroviruses.
Discussion BALB-and Harvey-MSV comprise a family of retroviruses whose mouse-and ratderived onc genes are closely related and whose 21,000-tool wt gene products have been causally implicated in their respective transforming functions (11,53,54). In the present report, we demonstrate that these viruses transform a novel hematopoietic target cell. Evidence that transformation was a direct result of BALB-and Harvey-MSV infection was derived from the one-hit titration pattern for bone marrow colony formation, the absence of any detectable effect of helper virus alone, and the ability to demonstrate the presence of sarcoma virus in cell lines established from such colonies. BALB-and Harvey-MSV induced colonies could be established in culture as continuous clonal lines with high proliferative capacity. Their ability to form rapidly growing tumors of donor origin and with the same hematopoietic blast cell morphology confirmed their malignant potential.
The hematopoietic blast cell transformants of BALB-and Harvey-MSV described in this report possessed none of the markers that identify mature cells within the known hematopoietic lineages. The low level of Fc receptors detected on some clones does not place them in any specific lineage or stage of maturation (45). Analysis of BALB-and Harvey-MSV transformants did not reveal any that contained detectable immunoglobulin or thymus antigen. In contrast, lymphoid cells transformed by Abelson-MuLV in tissue culture appear to be at an early stage of B cell differentiation. The majority of such transformants synthesize immunoglobulin heavy/z chain in the absence of light chain (47). In the avian system, reticuloendotheliosis virus (REV-T) transforms more immature lymphoid cells that generally lack immunoglobulin expression (55). Both Abelson-MuLV and REV-T lymphoid transformants have been reported to possess low or undetectable levels of TdT (56,55). In contrast, BALB-and Harvey-MSV transformed hematopoietic blast cell lines contained very high levels of this enzyme, comparable to that of normal thymus. The different phenotype of BALB-and Harvey-MSV transformants compared with that of Abelson-MuLVtransformed lymphoid cells induced under identical assay conditions demonstrates the exquisite specificity that retroviral transforming genes can have for cells at different stages of differentiation, even within the same lineage.
The growth-promoting actions of BALB-and Harvey-MSV for hematopoietic cells in culture is not limited to their transforming effects on early lymphoid cells reported here. These viruses also induce erythroid burst formation (16) and macrophage colony formation in infected bone marrow cell cultures (57). In neither case have permanent, malignant cell lines as yet been established. Thus, whether the growth-promoting activities of BALB-and Harvey-MSV on erythroid and macrophage cells is directly caused by virus transformation or the result of some indirect effect of the virus remains to be determined.
Infection of newborn mice with Harvey-MSV has been shown to result in erythroleukemia (15). We have confirmed the striking splenomegaly associated with Harvey-MSV infection and observed similar gross pathology in BALB-MSV inoculated animals. We have also observed tumor involvement of the thymic region in infected animals that has not been previously reported. When we analyzed bone marrow cells from such animals, we were able to detect hematopoietic colony-forming cells with the same blast cell phenotype observed after in vitro infection. In fact, the numbers of such colonies was only ~ 10-fold less than that observed with Abelson-MuLV. These findings indicate that the hematopoietic tumors induced by BALB-or Harvey-MSV may have a more complex etiology than has previously been supposed.
The occurrence of TdT in prelymphoid cells has been well documented (48,49). TdT is considered to be a specific marker for early lymphocytes because it is low or absent in mature lymphocytes. The existence of a TdT + Thy-1-bone marrow cell population has been indicated by findings that elimination of the Thy-1 + population by complement-mediated cytolysis does not significantly reduce TdT activity in the bone marrow (58). That some of Thy-1-TdT + bone marrow cells are committed to T cell differentiation has been indicated by the reduction in TdT activity after exposure to thymopoietin followed by anti-Thy-1 complement-mediated lysis (58). Our BALB-and Harvey-MSV transformants possessed a stable TdT + Thy-1-phenotype. Thus, these cells appear to represent the malignant counterpart of a normal lymphoid progenitor cell. It will be of interest to determine whether these cells have the capacity to differentiate along either T or B cell developmental pathways after transformation by temperature-sensitive mutants or exposure to agents that promote differentiation in other systems (25,(58)(59)(60). If so, these transformants should provide a useful approach for analysis of lymphoid cell ontogeny and the possible function of TdT in normal lymphocyte development.
Summary BALB-and Harvey-murine sarcoma viruses (MSV) comprise a family of retroviruses whose mouse-and rat-derived onc genes are closely related. These viruses induce sarcomas and erythroleukemias in susceptible animals. An in vitro colony assay that detects transformation of lymphoid cells by Abelson-murine leukemia virus was used to demonstrate that BALB-and Harvey-MSV transform a novel hematopoietic cell both in culture and in vivo. Bone marrow colony formation was sarcoma virus dependent, followed single-hit kinetics, and required the presence of mercaptoethanol in the agar medium. BALB-and Harvey-MSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The cells had a blast cell morphology and lacked detectable markers of mature cells within the myeloid or erythroid series. They also lacked detectable immunoglobulin /~ chain or Thy-1 antigen, markers normally associated with committed cells of the B and T lymphoid lineages, respectively. However, the transformants contained very high levels of terminal deoxynucleotidyl transferase (TdT), an enzyme believed to be specific to early stages within the lymphoid differentiation pathway. This phenotype distinguishes these BALB-and Harvey-MSV transformants from any previously reported hematopoietic targets of transforming retroviruses, including the pre-B lymphoid cell transformed by Abelson-MuLV under identical assay conditions. These newly identified lymphoid progenitor cell transformants may provide an important means of studying early stages of lymphoid ontogeny and the possible role of TdT in lymphoid development.