extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Oncogenic ras Causes Resistance to the Growth Inhibitor Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) in Breast Cancer Cells*

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation and promotes apoptosis in normal and malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of control. This inhibition appeared IGF-independent, since neither an IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant transformation of MCF-10A cells by transfection with Ha-rasoncogene abolished the inhibitory effect of IGFBP-3, concomitant with an increase in IGFBP-3 secretion and cell association of approximately 60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on IGFBP-3 secretion or cell association by ras-transfected or parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line that expresses activated Ha-ras, similarly has a high level of secreted and cell-associated IGFBP-3. In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IGFBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this level of inhibition was achieved with 100 ng/ml IGFBP-3. These results suggest that MAP kinase activation by oncogenic rasexpression causes IGFBP-3 resistance, a possible factor in the dysregulation of breast cancer cell growth.

Insulin-like growth factor binding protein-3 (IG-FBP-3) inhibits proliferation and promotes apoptosis in normal and malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of control. This inhibition appeared IGF-independent, since neither an IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant transformation of MCF-10A cells by transfection with Ha-ras oncogene abolished the inhibitory effect of IGFBP-3, concomitant with an increase in IGFBP-3 secretion and cell association of approximately 60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on IGFBP-3 secretion or cell association by ras-transfected or parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line that expresses activated Ha-ras, similarly has a high level of secreted and cell-associated IGFBP-3.
In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IG-FBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this level of inhibition was achieved with 100 ng/ml IGFBP-3. These results suggest that MAP kinase activation by oncogenic ras expression causes IGFBP-3 resistance, a possible factor in the dysregulation of breast cancer cell growth.
The potent growth promoting effects of the insulin-like growth factors (IGFs) 1 are regulated in all tissues by the six members of a family of structurally and functionally related proteins, the IGF-binding proteins (IGFBPs), which bind the IGFs with affinities of between 10 9 and 10 11 liters/mol (1). While sharing considerable amino acid homology, the IGFBPs undergo considerable post-translational modification to yield differently glycosylated, phosphorylated, and proteolytically cleaved products (2). IGFBP-3, a 45-kDa glycoprotein, is the predominant carrier of the growth factors in the circulation where, as a component of a 150-kDa complex consisting of IGFBP-3, IGF-I or -II, and the acid-labile subunit, it maintains a circulating reservoir of the IGFs, limits their insulin-like hypoglycemic potential, and helps to control their egress from the circulation to the extravascular tissues (3). In addition to its presence in the circulation, IGFBP-3 is found in the pericellular environment of many tissues, where it functions as a paracrine or autocrine modulator of the mitogenic effects of the IGFs.
There is now good evidence that IGFBP-3, a p53-inducible protein (4), inhibits proliferation and promotes apoptosis in normal and malignant cells by mechanisms that may be independent of its effects on IGF bioactivity (5)(6)(7)(8)(9)). An important role for IGFBP-3 in the modulation of breast cancer cell growth is suggested by the observation that DNA synthesis and cell proliferation in tumor-derived breast cell lines may be reduced by treatment with recombinant IGFBP-3 (7). In addition, the antiproliferative effects of agents as diverse as transforming growth factor-␤ (TGF-␤), retinoic acid, vitamin D analogs, and anti-estrogens is accompanied by induction of IGFBP-3 (6, 10 -12), and prevention of this induction with antisense oligonucleotides restores DNA synthesis. Very little is known of the mechanisms by which IGFBP-3 exerts these effects, however, and the factors involved in regulating cell sensitivity to IG-FBP-3 are completely uncharacterized. We now show that breast epithelial cells expressing a constitutively active Ras protein are resistant to IGFBP-3 and that abrogation of the MAPK/ERK signaling pathway restores sensitivity to IG-FBP-3. These findings implicate the Ras-MAPK/ERK pathway in the development of IGFBP-3 insensitivity in breast cancer.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture reagents and plasticware were from Trace Biosciences (North Ryde, New South Wales, Australia) and Nunc (Roskilde, Denmark). Bovine serum albumin (BSA), bovine insulin, hydrocortisone, epidermal growth factor (EGF), and protein A were purchased from Sigma. Cholera enterotoxin was purchased from ICN Biomedicals Australasia (Seven Hills, New South Wales, Australia). The MAP kinase inhibitor PD 98059, and monoclonal antibody against type 1 IGF receptor, ␣IR-3, were obtained from Calbiochem-Novabiochem (Alexandria, New South Wales, Australia). Polyclonal antibodies against Thr 202 /Tyr 204 phosphorylated and total p44/42 MAP kinase were from New England Biolabs (Beverley, MA). Recombinant human TGF-␤1 was purchased from Austral Biologicals (San Ramon, CA), and recombinant human IGF-I was the gift of Pharmacia and Upjohn (Stockholm, Sweden). Human IGFBP-3 was purified from Cohn fraction IV of human plasma and IGFBP-6 from SV-40-transformed fibroblastconditioned medium, as described previously (13,14). Electrophoresis reagents were purchased from Bio-Rad and Amrad-Pharmacia (Ryde, New South Wales, Australia). Protein A and IGF-I were radiolabeled with 125 I (ICN) using chloramine T.
[ 3 H]Thymidine Incorporation-For analysis of DNA synthesis, confluent cultures of cells in 24-well plates were changed to Hepes-buffered DMEM/F-12 containing 0.1% BSA for 48 h prior to addition of treatments. Spent media were replaced by fresh serum-free medium containing additives as indicated for individual experiments, and incubations were continued for 20 h. One Ci/well [ 3 H]thymidine (35 Ci/mmol, ICN) was added in 50 l of medium for a further 4-h incubation at 37°C. Monolayers were rinsed twice with ice-cold saline and fixed with 1 ml/well ice-cold methanol:acetic acid (3:1) at 4°C for a minimum of 2 h. Cells were solubilized in 0.5 ml of 5 g/liter of sodium dodecyl sulfate (SDS), and 250 l of each lysate mixed with scintillant (OptimaGold, Amersham Pharmacia Biotech) before counting for 2 min in a Hewlett-Packard ␤ counter.
Determination of Secreted and Cell-associated IGFBP-3-IGFBP-3 concentrations in conditioned media were determined by radioimmunoassay as previously reported (10). Cell-associated IGFBP-3 was assayed on intact monolayers using an immunological technique described in an earlier study (16). Briefly, spent media from cells treated in 24-well plates were removed for assay and replaced by 1 ml of fresh medium containing anti-IGFBP-3 serum or nonimmune serum (1:5000 dilution). After overnight incubation at 22°C, media were aspirated, and 1 ml of medium containing 20,000 cpm 125 I-labeled protein A was added for 2-3 h at 22°C. Cells were then washed twice with serum-free medium, solubilized with 5 g/liter SDS, and lysates counted for 2 min in a Hewlett-Packard ␥ counter. Cell-associated IGFBP-3 was determined from the total amount of radioactive protein A present in cell lysates and reflected the amount bound to IGFBP-3-antiserum complexes on the cells.
Statistics-All experiments were conducted in triplicate or quadruplicate at least twice; results shown are pooled data from all similar experiments unless otherwise indicated. Statistical significance was determined by analysis of variance and Fisher's PLSD on pooled data using Statview software for Macintosh (SAS Institute, Inc).

RESULTS
The MCF-10A cell line is a spontaneously immortalized, phenotypically normal breast epithelial line (17). When these cells were treated with human plasma-derived IGFBP-3 for 24 h, DNA synthesis (determined by incorporation of tritiated thymidine) was maximally inhibited to ϳ70% of control levels at 30 ng/ml IGFBP-3 (p Ͻ 0.01 compared with control), with a significant effect apparent with 10 ng/ml IGFBP-3 (p Ͻ 0.05) (Fig. 1A). This effect of IGFBP-3 was not due to the binding protein sequestering endogenous IGFs and inhibiting their mitogenic effects, because the monoclonal antibody ␣IR-3, which acts as an IGF antagonist at the type 1 IGF receptor (18), had no significant effect on basal DNA synthesis in MCF-10A cells (Fig. 1B); at the concentration used (5 g/ml) it was able to fully reverse stimulation by 5 ng/ml IGF-I (Fig. 1B). Similarly IG-FBP-6, which binds both IGF-I and IGF-II with high affinity, did not inhibit basal DNA synthesis when added at 100 ng/ml, but blocked the stimulatory effect of IGF-I (Fig. 1B).
MCF-10A cells transfected with the Ha-ras oncogene (MCF-10A ras ) exhibit characteristics of malignant transformation (15), including 2-3-fold increased DNA synthesis in the absence of added growth factors (not shown). By contrast with the MCF-10A cell line, MCF-10A ras cells appeared resistant to the inhibitory effects of IGFBP-3 (Fig. 1C), with concentrations up to 100 ng/ml IGFBP-3 failing to inhibit DNA synthesis in these cells. Although IGF signaling pathways might be expected to be activated in these cells, they showed no loss of sensitivity toward stimulation by exogenous IGF-I; on the contrary, IGF-I sensitivity was increased 4 -5-fold compared with MCF-10A cells (Fig. 1D).
As proteolysis of IGFBPs may decrease their bioactivity compared with their intact counterparts (19), we investigated whether the absence of IGFBP-3 inhibitory activity in MCF-10A ras cells was due to increased degradation of the protein.
Media conditioned by MCF-10A and MCF-10A ras cells treated with IGFBP-3 were analyzed by IGF ligand blot. By this method, increased proteolysis results in decreased signal attributable to intact 43-kDa IGFBP-3 (20), as the proteolyzed protein has reduced binding to iodo-IGF-I (21). As shown in Fig. 1E, there was no loss of 43-kDa IGFBP-3 in the MCF-10A ras cell-conditioned medium compared with the untransfected MCF-10A cells by ligand blot, confirming that the lack of an inhibitory effect of IGFBP-3 was not due to increased degradation of exogenous IGFBP-3 by the MCF-10A ras cells. MCF-10A ras cells also showed a similar sensitivity to the inhibitory effects of TGF-␤ as MCF-10A (Fig. 1F), indicating that resistance to IGFBP-3 was not due to a general change in sensitivity to inhibitory factors as a result of constitutive activation of the Ras signaling pathway. Taken together, these data indicate that the inhibition of DNA synthesis in breast epithelial cells by IGFBP-3 is not a simple consequence of IGF binding by IGFBP-3 and that expression of oncogenic ras by these cells results in resistance to inhibition by IGFBP-3.
Signaling via Ras involves the activation of intermediates in a number of downstream pathways (22); we therefore investigated whether sensitivity to IGFBP-3 in MCF-10A ras cells could be changed in the presence of specific inhibitors of these pathways. The inhibitor wortmannin (10 nM), which blocks the phosphatidylinositol 3-kinase pathway, had no effect on IG-FBP-3 sensitivity in MCF-10A or MCF-10A ras cells (not shown). However PD 98059, a synthetic flavone that acts as a specific inhibitor of activation of the MAPK/ERK (mitogenactivated protein kinase/extracellular signal-regulated kinase) kinase MEK (23) had a marked effect on IGFBP-3 sensitivity in ras-transfected cells. When exposed to 5 M PD 98059, each cell line showed 30 -40% inhibition of DNA synthesis over 24 h, an effect similar to that described in other cell types (23). Addition of 10 -100 ng/ml IGFBP-3 resulted in further inhibition of DNA synthesis ( Fig. 2A), implying that sensitivity to IGFBP-3 in these cells is restored when MEK activation is inhibited.
Increased signaling through Ras-dependent pathways, either as the result of mutations in ras itself as seen in a small proportion of breast tumors or via increased activity of factors that activate Ras, is believed to be an important contributing factor in the development and progression of breast malignancies (24). The tumor-derived Hs578T cell line expresses an activated Ha-ras gene (25), and we found that, like ras-transfected MCF-10A cells, Hs578T cells were relatively insensitive to growth inhibition by IGFBP-3, requiring 1000 ng/ml IG-FBP-3 for significant inhibition of DNA synthesis (Fig. 2B). These results indicate that in either transfected or tumorderived cell models, breast epithelial cells expressing constitutively active Ras are relatively resistant to the inhibitory effects of IGFBP-3. As was seen with the MCF-10A ras cells, however, co-incubation of Hs578T cells with PD 98059 (5 M) and IGFBP-3 at a concentration of 100 ng/ml resulted in significant inhibition of DNA synthesis (Fig. 2B), indicating a 10-fold increase in sensitivity compared with IGFBP-3 treatment in the absence of PD 98059. To confirm that this reagent was inhibiting MAP kinase phosphorylation in the MCF-10A ras and Hs578T cells, lysates from cells treated for 24 h with PD 98059 were analyzed by immunoblot using antibodies against phosphorylated p44/42 MAPK (Fig. 2C). Incubation with 5 or 20 M PD 98059 reduced phosphorylated MAPK by 50 -60% (determined by densitometric scanning of the autoradiograms shown), in the absence of any change in total MAPK. Thus, partial blockade of Ras signaling by inhibiting MAPK/ERK activation restores sensitivity to IGFBP-3 in breast cells expressing oncogenic ras.
One possible mechanism by which PD 98059 might increase sensitivity to inhibition by IGFBP-3 would be through increasing the responsiveness to endogenous IGFs. However, in the presence of PD 98059, neither ␣IR-3 nor IGFBP-6 caused further inhibition of DNA synthesis in MCF-10A ras or Hs578T cells (not shown), indicating that restoration of IGFBP-3 inhibitory activity was unrelated to its binding of endogenous IGFs. Furthermore, Hs578T cells, which show no response to exogenous IGF-I (27), did not show increased IGF sensitivity in the presence of PD 98059 (not shown).
It has been proposed that the intracellular actions of IG-FBP-3 are initiated through its interaction with a cell-surface receptor (26), although no signaling receptor has been identified. In common with other peptide receptors, the cell-surface expression of such a receptor might be down-regulated by a high concentration of its ligand. In contrast with many other breast cancer cells which secrete little or no IGFBP-3 (10), Hs578T cells secrete relatively large amounts of IGFBP-3, and ras transfected MCF-10A cells similarly secreted higher levels than their nontransfected counterparts (Table I). To determine whether the restoration of sensitivity to IGFBP-3 in response to PD 98059 was the result of decreased production of IGFBP-3, and thereby release from down-regulation of a putative signaling receptor, IGFBP-3 concentrations in medium conditioned by cells treated with PD 98059 were measured. As shown in  Table I, PD 98059 treatment, while restoring sensitivity to exogenous IGFBP-3, had no significant effect on IGFBP-3 secretion in any of the cell lines tested.
Cell association of IGFBP-3 has been shown in some breast cancer cell lines to correlate with its growth inhibitory effect (26). We therefore investigated whether the amount of cellassociated IGFBP-3 changed under conditions where sensitivity to its inhibitory effects was increased. In the absence of IGFBP-3 or PD 98059, cell-associated IGFBP-3 levels were similar in the MCF-10A ras and Hs578T cells and 2-3-fold higher than in either the parent or control vector-transfected MCF-10A cell lines (data not shown). As shown in Fig. 3A for MCF-10A ras cells, addition of IGFBP-3 significantly increased the amount of IGFBP-3 associated with the cell at all doses tested. However, co-addition of 5 M PD 98059 had no effect on cell-associated protein in the absence or presence of concentrations of IGFBP-3 shown previously (in Fig. 2) to inhibit DNA synthesis. In the Hs578T cells, a significant increase in the amount of cell-associated IGFBP-3 was seen with 100 ng/ml dose of exogenous IGFBP-3 only, and this did not change in the presence of PD 98059 (Fig. 3B). These observations argue against a direct link between the degree of cell association of IGFBP-3 and its inhibition of DNA synthesis.

DISCUSSION
Although antiproliferative and pro-apoptotic effects of IG-FBP-3 have been reported in a variety of breast cancer cell lines, its actions in nonmalignant breast cells have not been described previously. The data presented in this report indicate that normal breast epithelial cells are considerably more sensitive to IGFBP-3 than tumor-derived breast cancer cells, which require 500 -1500 ng/ml IGFBP-3 to achieve a similar level of inhibition to that seen with 10 -100 ng/ml in the nontransformed cells (7,27). Our findings also indicate that resistance to IGFBP-3 is induced in breast cells transfected with oncogenic ras, thereby implicating increased activity of Ras-dependent signaling pathways in the development of IGFBP-3 insensitivity. In contrast, sensitivity toward the growth inhibitor TGF-␤ was unaffected by ras transfection, suggesting some specificity in the IGFBP-3 effect.
Activation of the MAPK/ERK pathway, whether by ras mutation or other mechanisms such as growth factor receptor activation, may be a common feature of the malignant growth of breast epithelial cells (24), and our results would indicate that in these situations resistance to the tumor-suppressive activity of endogenous IGFBP-3 would be one result of such activation. Several lines of evidence indicate that changes in sensitivity to IGFBP-3 resulting from oncogenic ras expression or MAPK blockade are unrelated to effects on IGF signaling or sensitivity. First, MCF-10A cells are not inhibited by IGFBP-6 or the type 1 IGF receptor antagonist, ␣IR-3, despite their sensitivity to IGFBP-3. Second, transfection of MCF-10A cells with oncogenic ras actually increased their sensitivity to IGF-I, so that loss of IGF-I responsiveness could not explain their loss of inhibition by IGFBP-3. Third, the insensitivity of Hs578T cells to IGF-I is not altered in the presence of PD 98059.
Increased proteolysis of exogenous IGFBP-3 in the MCF-   10A ras cell line was also investigated as a possible cause of the lack of response to its inhibitory effects. In other cell systems, limited degradation of IGFBP-3 has been demonstrated to affect its IGF-independent (28) and -dependent (29) actions; however, there was no significant loss of intact IGFBP-3 over the 24-h treatment period for the MCF-10A ras cells. We also investigated the possibility that a high level of endogenous IGFBP-3, which is characteristic of both the Hs578T and MCF-10A ras cell lines, was leading to down-regulation of a putative signaling receptor, but found that sensitivity to IGFBP-3 could be reinstated in the absence of any change in the level of secreted IGFBP-3.
It has been reported that the antiproliferative effects of IG-FBP-3 correlate with its presence on the cell surface of the cell (27). This observation has been interpreted as evidence for the existence of a signaling receptor (26), which has been suggested recently to be the type V TGF-␤ receptor (30) despite a lack of specific evidence that this receptor mediates either TGF-␤ or IGFBP-3-dependent events. Although we identified IGFBP-3 in association with the surface of MCF-10A and MCF-10A ras cells, its concentration did not correlate with sensitivity to its effects. In IGFBP-3-insensitive MCF-10A ras cells, addition of IGFBP-3 resulted in an increase in the amount of cell-associated IG-FBP-3 without inhibiting DNA synthesis, while treatment with PD 98059, which restored sensitivity to the growth inhibitory effects of IGFBP-3 in both Hs578T and MCF-10A ras cells, did not alter the amount of cell-associated IGFBP-3. This suggests that cell binding of IGFBP-3 per se does not lead to inhibition of DNA synthesis and implies the existence of other factors, either intracellular or extracellular, involved in the regulation of IGFBP-3 inhibitory signaling. Our findings would also indicate that the expression or activity of such factors is Ras-dependent.
We showed recently that in T47D breast cancer cells transfected to overexpress IGFBP-3, the cell population changed over several passages from a state of relative cell cycle arrest by IGFBP-3 to a state of insensitivity to its inhibitory effect, where cell growth was uninhibited by high levels of secreted and cell-bound IGFBP-3 (31). Although the mechanism for this transition is unknown, we have now demonstrated that the expression of oncogenic ras, either in a transfected cell line or in breast tumor-derived cells, is associated with resistance to IGFBP-3 inhibitory signaling. IGFBP-3 is abundant in the human circulation (32), where a high concentration is a negative risk factor for the development of breast and prostate cancer (33,34). We propose that the development of resistance to IGFBP-3 may be a key step in the dysregulation of breast cancer cell growth, raising the possibility that future therapies could be directed toward maintaining or restoring sensitivity to the growth inhibitory effects of this protein.


extfieldsofstudy: Medicine Biology

text:
The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity.

The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3–dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)–like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)–treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non– 5-FU–treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.

T he BCR/ABL fusion oncogene, the product of the t(9;22) Philadelphia chromosome (Ph), 1 exists in three principal forms (P190, P210, and P230) that arise from dis-tinct breakpoints in the BCR gene on chromosome 22, resulting in translocation of BCR exon 1 (1), exons 1-12/13 (2), or exons 1-19 (3), respectively, to the c-ABL gene on chromosome 9. These different oncogenes give rise to three distinct fusion proteins of molecular mass 190, 210, and 230 kD, which contain the same portion of the c-Abl tyrosine kinase in the COOH terminus but include different amounts of Bcr sequence at the NH 2 terminus.
In humans, there is evidence that the three different forms of BCR/ABL are associated with distinct forms of leukemia (for reviews, see references 4 and 5). The P210 form of BCR/ABL is found in hematopoietic cells of patients with chronic myeloid leukemia (CML) in stable phase, and in acute lymphoid and myeloid leukemias (6)(7)(8), although some patients with acute leukemia and P210 are likely to be cases of CML diagnosed in blast crisis. In contrast, the P190 form of BCR/ABL is commonly found in Ph-positive acute B lymphoid leukemia (ALL [9]) and occasionally in acute myeloid leukemia (10), but is rarely if ever observed in CML (11)(12)(13). Recently, several patients were described who had a Ph-positive myeloproliferative syndrome resembling CML but with very mild clinical symptoms, including a lower peripheral blood white cell count consisting principally of neutrophils, less anemia, less severe splenomegaly, and a delayed or absent transformation to blast crisis (14). These patients were found to have the BCR exon 19/ ABL exon 2 (e19/a2) junction characteristic of P230 BCR/ABL . It was proposed that patients with the e19/a2 fusion comprise a distinct clinical entity called neutrophilic CML, with a much more benign clinical course than that associated with the traditional b2/a2 or b3/a2 P210 BCR/ ABL fusion. However, several subsequent reports described patients with the e19/a2 fusion and typical CML (15)(16)(17). These observations raise the question of whether different forms of Bcr/Abl protein have intrinsically different leukemogenic activity in hematopoietic cells.
Consideration of the primary structures of P190, P210, and P230 indicates that P210 and P230 contain several potential functional motifs encoded by the BCR portion of the fusion gene (Cdc24/Dbl and plekstrin homology domains and a portion of a RacGAP catalytic domain, respectively) that might plausibly alter the oncogenic activity of these larger proteins relative to P190. Indeed, characterization of the tyrosine kinase and in vitro transformation activities of P190 and P210 Bcr/Abl has suggested that these proteins have distinct transforming properties. In an immune complex kinase assay, P190 has higher tyrosine kinase than P210, with P190 and P210 activity both elevated relative to c-Abl (18,19). P190 is more efficient than P210 in transformation of Rat-1 fibroblasts, as assessed by colony formation in soft agar (18). Both P210 and P190 BCR/ABL can stimulate the growth of primary B cells in long-term in vitro cultures, eventually leading to oligoclonal populations of pre-B cells that are independent of stroma and leukemogenic in syngeneic mice (20). P190 is more potent than P210 in this growth-stimulatory assay, with P190-transduced cultures reaching high cell density and stromal independence more frequently and sooner than with P210 (21). The mechanism of increased growth stimulation of B lymphoid cells by P190 is not known. Most studies of P190 and P210 have found similar sets of tyrosine-phosphorylated proteins in cells transformed by the two oncogenes (22). A notable exception is signal transducer and activator of transcription STAT6, a transcription factor normally activated by IL-4 and implicated in lymphoid proliferative responses (23) that is preferentially tyrosine phosphorylated and activated by P190 but not P210 Bcr/Abl (24). The only model system where Bcr/Abl induces a myeloproliferative syndrome closely resembling human CML is retroviral transduction of mouse bone marrow in vitro, followed by transplantation of transduced cells into irradiated syngeneic recipients (for a review, see reference 25). In this assay, the P210 form of BCR/ABL induced a fatal CMLlike syndrome with massive elevation of neutrophils and maturing myeloid cells in peripheral blood, marrow, spleen, and liver (26)(27)(28)(29). In addition to the CML-like syndrome, other transplanted mice developed leukemias of B lymphoid, monocyte/macrophage, T lymphoid, or erythroid lineages (26,30). Because these mice develop leukemias that closely resemble several human Ph-positive leukemias, the bone marrow transduction/transplant model is perhaps the best system for directly comparing the leukemogenic activity of different forms of BCR/ABL . However, as originally described, the system was hampered by difficulty in generating high-titer replication-defective BCR/ABL retrovirus stocks, leading to a low efficiency of generation of diseased animals. Under the best circumstances, the CML-like syndrome was observed in only ف 25% of transplant recipients, and some transplanted mice did not develop any malignancies (26). Despite these difficulties, P190 was observed to induce a similar CML-like syndrome in mice at low frequency (31). In experiments which used replication-competent Moloney murine leukemia helper virus to increase transduction efficiency, P190 was also observed to induce myeloid leukemias with excess neutrophils, suggestive of CML (32). In addition, the latency of P190-induced myeloid and lymphoid leukemias after transplant was shorter than for P210, suggesting that P190 was more potent in the induction of both types of leukemia.
With modifications to the original bone marrow transduction protocol, we and others (33) have recently been able to obtain 100% incidence of the CML-like syndrome in mice transplanted with P210 BCR/ABL -transduced marrow, allowing this method to be used as an assay for the first time. Here, we report a direct comparison of the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL .

Materials and Methods
DNA Constructs. A cDNA encoding the P230 form of BCR/ ABL was generated by a PCR strategy. A 5 Ј primer containing the AatII site at nucleotide 3184 in the BCR cDNA open reading frame and the adjacent e19/a2 BCR/ABL junction sequence and a 3 Ј primer containing the KpnI site at nucleotide 744 of the human c-ABL type Ia cDNA were used to generate a 307-bp PCR product from AatII to KpnI, spanning the P230 BCR/ABL junction. The product was subcloned and completely sequenced to verify the correct reading frame and lack of other mutations, then used to generate a complete P230 cDNA. The P190, P210, and P230 BCR/ABL cDNAs were introduced as EcoRI fragments into the unique EcoRI site of the murine stem cell virus (MSCV)based retroviral vector, MSCVneoEB (34).
In Vitro Kinase Assay. Immunoprecipitation of Abl proteins from transfected 293 cells or transduced Ba/F3 cells and immune complex kinase assay was as described previously (19,35), except that a glutathione S -transferase (GST)-c-Crk 120-225 fusion protein was used as an exogenous substrate. To normalize for the amount of Abl proteins, cells were labeled for 12 h with [ 35 S] l -methionine before harvesting. The amount of 32 P incorporation was quantitated by PhosphorImager analysis (STORM 850; Molecular Dynamics), whereas 35 S incorporation was quantitated by digital camera and analysis by NIH Image v1.59 software. Relative protein levels, which were within 15% of one another, were corrected for methionine content and used to normalize 32 P incorporation to calculate relative kinase activity.
Generation of Retrovirus Stocks. All DNAs were purified by two rounds of buoyant density centrifugation in CsCl. 10 g of retroviral vector DNA and 5 g of MCV-ecopac, an ecotropic single-genome packaging construct (36), were transfected per 6-cm dish of 293T cl. 17 cells (37), as described (36). Medium was changed at 24 h, and virus supernatant was harvested at 48 h after transfection. Supernatant was passed through a 0.45-filter, aliquoted, and frozen at Ϫ 80 Њ C. An aliquot was thawed, the virus titer for neomycin resistance was determined by transduction of NIH 3T3 cells, and a screen for the presence of replication-competent helper virus was carried out with a lacZ -3T3 indicator cell line. All viruses had titers of 3.5-4.0 ϫ 10 6 neomycin-resistant CFU/ml, passed the correct proviral structure by Southern blot analysis, and were free of detectable helper virus activity.
Transformation of Cytokine-dependent Hematopoietic Cell Lines. 32D cl3 cells and Ba/F3 cells were grown, transduced, and selected for G418 resistance and IL-3 independence as described previously (19). At 96 h after transduction, viable cells were counted and plated at 4 ϫ 10 4 cells in 1.0 ml RPMI medium without IL-3 and neomycin in triplicate wells of a 24-well plate. Cells were incubated at 37 Њ C, and the viable cell count was determined daily for 3 d.
Bone Marrow Transduction and Transplantation. All animal studies were approved by the Animal Use and Care Committees of the Center for Blood Research and Harvard Medical School. Balb/c mice (The Jackson Laboratory or Taconic Farms) from 6 to 12 wk of age were used in all experiments. In some experiments, male donor mice were primed by intravenous injection with 5-fluorouracil (5-FU; 200 mg/kg) 4 d before harvest. Male donors were killed by CO 2 asphyxiation, femur and tibia were collected, and bone marrow was harvested by flushing with syringe and 26-gauge needle. Cells were counted and plated without removal of erythrocytes at 2 ϫ 10 7 cells per 10-cm plate in prestimulation medium (38) of DME, 15% (vol/vol) inactivated FCS, 5% (vol/vol) WEHI-3B conditioned medium, penicillin/ streptomycin, 1.0 g/ml ciprofloxacin, 200 M l -glutamine, 6 ng/ml recombinant murine IL-3 (Genzyme), 10 ng/ml recombinant murine IL-6 (Genzyme), and 50-100 ng/ml recombinant murine stem cell factor (SCF; PeproTech). With non-5-FU-treated marrow, 10 ng/ml recombinant murine IL-7 (Genzyme) was also included. After prestimulation for 24 h at 37 Њ C, viable cells were counted and transduced with retroviral stocks in the same medium containing 50% retroviral supernatant, 10 mM Hepes, pH 7.4, and 2 g/ml polybrene. To increase transduction efficiency (39), virus and cells were cosedimented at 1,000 g for 90 min in a Sorvall RT-6000 centrifuge. Medium was changed after a 2-4-h adsorption period. At 48 h, a second round of transduction and cosedimentation was performed, and the cells were collected 2 h later, washed once in HBSS, and counted. Recipient female mice were prepared by two doses of 450-cGy gamma irradiation separated by 3 h. Transduced marrow cells were transplanted by injection of 0.2-0.5 ϫ 10 6 cells (5-FU-treated marrow) or 1.0 ϫ 10 6 cells (non-5-FU-treated marrow) in 0.4-0.5 ml HBSS into the lateral tail vein. After transplant, recipients were housed in microisolator cages supplied with acidified (pH 2.0) water.
Analysis of Diseased Mice. After transplant, recipient mice were evaluated daily for signs of morbidity, weight loss, failure to thrive, and splenomegaly. Premorbid animals were killed by CO 2 asphyxiation, peripheral blood was obtained from the retroorbital venous plexus, and hematopoietic tissues were removed. Depending on the individual animal, hematopoietic tissues and cells were used for several applications, including histopathology, in vitro culture, FACS ® analysis, secondary transplantation, genomic DNA preparation, protein lysate preparation, or lineage analysis (see below). The clinical features and histopathology of BCR/ ABL -induced CML-like disease, B lymphoid leukemia, and macrophage tumors were very similar to those observed previously (26).
Lineage Analysis. Peripheral blood samples generally served as a nearly pure source of neutrophils and their precursors; on occasion, neutrophils were purified further by positive selection for Ly-6G (Gr-1) antigen expression by immunomagnetic beads (MicroMACS ® ; Miltenyi Biotec). Macrophages were isolated from peritoneal washes, bone marrow, liver, or spleen by adherence and often subsequent culture on bacterial petri dishes in the presence of L929-conditioned medium as a source of CSF-1. Erythroid cells were purified from spleen by sedimentation through Ficoll-hypaque or by positive selection with TER119 mAb and immunomagnetic beads. B lymphoid cells were purified from pooled lymph nodes or spleen by positive selection with anti-B220 mAb, and T cells were purified by isolation of thymocytes or by positive selection of spleen or peripheral blood with anti-Thy-1.2 mAb. In all cases, the purity of the population was assessed by Wright-Giemsa staining of a cytospin specimen, and only those samples with at least 80% purity as judged from cell morphology were used for Southern blot analysis. Genomic DNA was prepared from each population, digested with BglII, and hybridized with a radioactive probe from the proviral neomycin resistance gene to determine the number of distinct proviral integrations in each sample. Subsequently, the blots were stripped and reprobed with a radioactive probe from the human c-ABL gene that detects a common 2.2-kb fragment from all proviruses, allowing determination of the total proviral content of each hematopoietic lineage. To control for differences in DNA loading, the intensity of hybridization of a fragment from the endogenous mouse c-abl gene was used as an internal control. Intensity of hybridization was determined by PhosphorImager analysis (Molecular Dynamics), and the ratio of intensity of hybridization of the BCR/ABL fragment to murine c-abl fragment was compared with that of genomic DNA standards containing a single proviral copy, with the results expressed as proviral copy number per diploid genome. A proviral copy number of 0.2 or less is consistent with the absence of provirus from a particular hematopoietic lineage.
Secondary Transplants and Day 12 Spleen Colony Assay. For adoptive transfer of the CML-like syndrome, 1-2 ϫ 10 6 bone marrow and/or splenocytes from primary animals were injected intravenously into sublethally irradiated (450 cGy) female Balb/c recipient mice. For isolation of day 12 spleen colonies, pairs of lethally irradiated (900 cGy) Balb/c mice were injected intravenously with 3 ϫ 10 3 , 1 ϫ 10 4 , and 3 ϫ 10 4 nucleated marrow cells from primary animals. Recipients were killed 12 d later, spleens were isolated, and macroscopic colonies were dissected out with the aid of a stereoscopic microscope. For those colonies not visibly red in appearance, cytospin preparations were examined by Wright-Giemsa staining to ensure the mixed myeloid origin of the colony. Genomic DNA was then prepared and analyzed as described above.
Different Leukemogenic Activity of P190, P210, and P230 BCR/ABL Western Blot Analysis. Protein lysates were prepared from peripheral blood myeloid cells by resuspension of 10 7 cells in 75 l RIPA buffer with immediate addition of an equal volume of 95 Њ C 2 ϫ sample buffer and boiling for 10 min. Amounts of protein were standardized by SDS-PAGE and Coomassie blue staining. From other hematopoietic tissues and cultures, cell lysates in RIPA were quantitated by Bio-Rad protein assay. SDS-PAGE and Western blotting with anti-Abl (8E9; PharMingen) and antiphosphotyrosine (4G10; Upstate Biotechnology, Inc.) antibodies were performed as described (19).

Results
P230 Has Lower In Vitro Kinase Activity Than P210 and P190. Previous studies indicated that P190 Bcr/Abl had intrinsically higher tyrosine kinase activity, measured as autophosphorylation or phosphorylation of an exogenous substrate, than did P210, while both were higher than c-Abl (18,19). In a direct comparison after immunoprecipitation from transfected 293 cells, we again confirmed this and found a significantly lower kinase activity for P230 Bcr/Abl (Fig. 1). Corrected for levels of Abl protein, P190 had 7-fold increased phosphorylation of a GST-Crk substrate relative to c-Abl, whereas P210 and P230 had 5.4-and 3.7-fold increased activity, respectively, with similar results for autophosphorylation. There was no difference in the kinase activity of c-Abl and the oncogenic SH3-deleted c-Abl protein, as observed previously (40). Similar results were obtained when the Bcr/Abl proteins were immunoprecipitated from stably transformed Ba/F3 cells (see below; data not shown).
The Three Forms of BCR/ABL Induce Different Proliferative Responses in Cultured Lymphoid Cells. To compare the transforming ability of the three BCR/ABL oncogenes in cultured hematopoietic cells, we introduced them by retroviral transduction into IL-3-dependent myeloid (32D cl3) and lymphoid (Ba/F3) cell lines. P210 BCR/ABL has been previously demonstrated to transform each cell line to become independent of IL-3 for survival and growth (41, 42) by a mechanism that does not involve autocrine production of growth factors. To avoid any differences due to prolonged culture in the presence of BCR/ABL, populations of transduced cells were selected in neomycin and immediately deprived of IL-3, with measurement of cell survival and proliferation within 96 h of transduction. With both cell types, populations of cells that were independent of IL-3 for survival and growth were selected with equal efficiency after transduction with all three forms of BCR/ABL, but not with the parental MSCVneoEB virus, demonstrating that P190, P210, and P230 are all capable of transforming myeloid and lymphoid cells to cytokine independence (Fig. 2). There was no difference in the growth rate of 32D cells transduced with any of the three forms of BCR/ABL in the absence of IL-3 or of vector-transduced cells in the presence of IL-3 ( Fig. 2 A). In contrast, BCR/ABL-transduced Ba/F3 cells in the absence of IL-3 proliferated more slowly than vector-transduced cells in the presence of IL-3, with P190transduced cells showing the highest proliferation rate, followed by P210 and then P230 (Fig. 2 B). The level of expression of the three Bcr/Abl proteins and the spectrum of tyrosine phosphorylated proteins were similar within each of the two cell types (data not shown). These results suggest that the three forms of BCR/ABL induce an identical proliferative response in myeloid factor-dependent cells, but deliver a submaximal growth stimulus in lymphoid cells that parallels their intrinsic tyrosine kinase activity.
All Three Forms of BCR/ABL Induce an Identical CML-like Syndrome in Mice. To compare directly the ability of the three forms of BCR/ABL to induce a myeloproliferative syndrome in mice, we used the bone marrow transduction/transplantation model system (26), with several modifications to the original protocol designed principally to increase transduction efficiency (see Materials and Methods). With the modified protocol, we observed that all mice transplanted with bone marrow transduced with each of the three forms of BCR/ABL developed a fatal CML-like myeloproliferative syndrome within 4 wk after transplantation when marrow from 5-FU-treated donors was used (Fig. 3). The disease was characterized principally by massive expansion of maturing myeloid cells in bone marrow, spleen, liver, and peripheral blood. The peripheral blood leukocyte count at death was 2-4 ϫ 10 5 /l, composed predominantly of mature neutrophils, along with metamyelocytes, myelocytes, and promyelocytes, indicative of a shift towards less differentiated myeloid cells. The spleen and liver were greatly enlarged and disrupted by large numbers of maturing myeloid cells from the neutrophil lineage, along with significant erythropoiesis and increased megakaryocytes. Nearly all mice with the CML-like syndrome also had small to medium-sized collections of macrophages centered on portal areas in the liver. In addition, all animals had focal consolidation of the lungs with maturing myeloid cells and extensive intraparenchymal hemorrhage, which may have been the ultimate cause of death of these animals. Bone marrow showed increased cellularity with maturation and an increased myeloid to erythroid ratio, while lymph nodes and thymus were normal. In most cases, the CMLlike syndrome could be efficiently transferred to secondary recipients by injection of bone marrow (data not shown), but as previously reported (43), for some primary animals the disease was not readily transplanted.
There was no significant difference in the histopathology, average survival, peripheral blood leukocyte count, or spleen weight of the disease induced by the three forms of BCR/ABL (Fig. 3 insert, and data not shown). Because the spleen is the main reservoir of myeloid cells in these diseased mice, this implies that mice transplanted with P190-, P210-, and P230-transduced marrow have identical total myeloid cell burdens at death, indicating that the proliferative stimulus induced in myeloid cells by the three BCR/  (Insert) Similar myeloid cell levels in mice with CML-like disease induced by the three forms of BCR/ABL. Average Ϯ SE (bars) of peripheral blood leukocyte count (ϫ10 3 / l, left) and spleen weight (in grams, right) for the three different BCR/ABL genotypes. The difference in mean survival of P190-transduced versus P210-transduced mice was significant at P ϭ 0.1 but not at P ϭ 0.05 (t test); none of the other pairwise differences in survival, leukocyte count, or spleen weight were significant (P ϭ 0.1). BMT, bone marrow transplantation.
ABL oncogenes is very similar. Although human P190 ϩ CMLs may be characterized by monocytosis (13), the percentage of monocytes in peripheral blood of mice with CML-like disease was Ͻ10% and did not differ significantly between BCR/ABL genotypes (data not shown).
The CML-like Syndrome Is Polyclonal and Involves Several Hematopoietic Lineages, Suggesting a Primitive Multipotential Target Cell. Mice with the CML-like syndrome were analyzed for the presence of Bcr/Abl protein by Western blot and for the BCR/ABL provirus by Southern blot of genomic DNA. Bcr/Abl protein was detected in peripheral blood leukocytes (principally neutrophils and their precursors; Fig.  4 A) and in peritoneal macrophages (Fig. 4 B), confirming the expression of the BCR/ABL provirus in two distinct myeloid lineages. To identify the BCR/ABL provirus, genomic DNA from total spleen, liver, peripheral blood, or bone marrow was digested with XbaI (which cuts once in each proviral LTR) or BglII (which cuts once in proviral DNA sequences 3Ј of the BCR/ABL cDNA insert) and probed with a radioactive fragment from the neomycin resistance gene. The XbaI digest indicated that these tissues contained the BCR/ABL provirus at about one proviral copy per cell (Fig. 5), confirming that the increased myeloid cells in these animals were a primary part of the malignant process and not a secondary or reactive phenomenon (30,44). The BglII digest, which yields a distinct hybridizing fragment for each unique proviral integration site, demonstrated that multiple independent clones contributed to the myeloid cell ex-pansion in these mice (Fig. 5), with 4-12 clones (average 9) observed in recipients of P190-, P210-, and P230-transduced marrow. Interestingly, the relative abundance of some clones differed between DNA samples from the same animal, suggesting a variable contribution of individual clones to myelopoiesis in different tissues. The presence of polyclonal disease suggests that the presence of BCR/ABL alone is sufficient to induce the CML-like syndrome.
To determine which hematopoietic lineages were involved in the malignant process, we performed lineage analysis by purifying different hematopoietic cell populations and determining the BCR/ABL proviral status by Southern blot. With all three oncogenes, mice with the CML-like syndrome carried the BCR/ABL provirus at or above single-copy levels in neutrophils, macrophages, erythroid cells, and splenic B lymphoid cells, and in some cases peripheral node lymphocytes and thymocytes (Fig. 6, A-C). Most of the independent proviral clones present were found in each lineage. These data indicate transduction of a bone marrow target cell capable of differentiation via multiple myeloid and lymphoid pathways, suggesting that the BCR/ABL target cell for the CML-like syndrome is a multipotential progenitor cell.
To further define the nature of this target cell, we performed secondary transplant experiments to isolate day 12 spleen colonies derived from selected primary mice with the CML-like syndrome. Such colonies are clonal, of mixed myeloid origin, have limited self-renewal potential, and are de- rived from a cell (CFU-S 12 ) with characteristics of an early multipotential progenitor with some stem cell-like properties (45). It was previously shown (26) that an animal with the CML-like syndrome carried the retroviral provirus in the majority of secondary day 12 spleen colonies, suggesting that the target cell for the CML-like syndrome was an early progenitor that could generate CFU-S 12 . In this study, we also detected the BCR/ABL provirus in the majority of day 12 spleen colonies in secondary transplants from mice with the CML-like syndrome (Fig. 6, A, B, and D). Collectively, in 10 primary animals with the CML-like syndrome, 64% (28 out of 44) of day 12 spleen colonies from secondary transplants carried the BCR/ABL provirus (Fig. 6, and data not shown). Interestingly, of the many distinct proviral clones present in the bone marrow of the primary animals, a small subset of clones was detected frequently or exclusively in secondary day 12 spleen colonies. For example, two minor clones were found in four out of seven colonies derived from an animal with P190-induced CML-like disease (Fig. 6 A), whereas a single minor clone generated seven out of eight colonies from a mouse with P210-induced disease (Fig. 6 D).
We observed a similar phenomenon in secondary recipients that developed the CML-like syndrome after transplantation of larger numbers of marrow cells. Instead of developing polyclonal disease with the same spectrum of proviral clones as the primary animal, myeloid cells from secondary mice with CML-like disease typically contained only a single minor clone from the donor (Fig. 6, C and D). In one animal, the same clone that contributed exclusively to day 12 spleen colonies was also recovered from secondary mice with CML-like disease (Fig. 6 D). These findings suggest that the target cell for induction of the CML-like syndrome is heterogeneous, with all clones exhibiting multilineage repopulating ability but only some clones capable of efficient generation of CFU-S 12 and secondary disease.
P190 BCR/ABL Induces More Aggressive B Lymphoid Leukemia Than P210 or P230 When Non-5-FU-treated Marrow Is Used for Transduction. Because animals with the CMLlike syndrome die within 4 wk of transplantation, it is not possible to assess the incidence or severity of BCR/ABLinduced hematologic malignancies of longer latency or duration in this system. Multipotential myeloid target cells for BCR/ABL are enriched for by 5-FU treatment (46), whereas the target cell for Abelson virus-induced pre-B leukemia is fairly abundant in normal marrow from Balb/c mice (47). Therefore, we reasoned that the use of bone marrow from donors that had not been pretreated with 5-FU might allow us to observe the characteristics of other leukemias induced by BCR/ABL, including lymphoid leukemia.
Indeed, when marrow from non-5-FU-treated donors was used for retroviral transduction, we observed three distinct hematopoietic neoplasms with each of the three forms of BCR/ABL (Fig. 7). We observed the CML-like syndrome in about half of transplant recipients. The survival of mice with the CML-like syndrome induced by transduction of non-5-FU marrow (21-49 d) was somewhat longer than for recipients of 5-FU-treated marrow. Otherwise, the syndrome appeared pathologically identical to that observed with 5-FU-treated marrow, with increased neutrophil counts, hepatosplenomegaly, and pulmonary hemorrhage. Analysis of proviral integration in myeloid cells showed that significantly fewer (one to three) independent proviral clones contributed to the CML-like disease in these mice, compared with recipients of transduced marrow from 5-FU-treated donors (Fig. 5). Lineage analysis of selected mice demonstrated the same proviral clone(s) in neutrophils, macrophages, erythroid cells, and B lymphoid cells (data not shown), indicating the target cell for the CML-like disease in normal marrow, like that from 5-FUtreated marrow, had multilineage differentiation potential.
The other animals developed acute leukemia of B lymphoid type, or tumors of monocyte/macrophages. Animals with B lymphoid leukemia exhibited modest splenomegaly (0.2-0.4 g) and lymphadenopathy with infiltration with lymphoblasts, and a bloody pleural effusion, containing high levels of malignant lymphoid cells, that appeared to be the cause of death. The lymphoblasts expressed high levels of Bcr/Abl protein (Fig. 4 C), were negative for myeloid and T lymphoid cell surface markers, but positive for CD45R (B220), CD43, 6C3/BP-1, and CD24 (data not shown), indicating an immature B cell phenotype. The lymphoid leukemias were efficiently transplanted to secondary recipients, with animals receiving 3 ϫ 10 6 tumor cells from lymph node or pleural effusion succumbing to an identical disease within 4-5 wk. Animals with the monocyte/macrophage tumors presented with enlargement of the liver and infiltration of periportal areas with tumors of cells with large vacuolated cyto-plasm; similar tumors were often found in the spleen and mesentery, and most animals had a prominent ascites composed exclusively of macrophages. The tumor cells expressed high levels of Bcr/Abl protein (Fig. 4 B) and were positive for the cell surface markers Mac-1 and F4/80 but negative for lymphoid markers (data not shown). Some mice exhibited characteristics of two diseases simultaneously (Fig. 7); because of the distinct and very uniform features of the three Figure 6. The target cell for the CML-like disease has multilineage repopulating ability but is heterogeneous for self-renewal as assessed by secondary transplantation. (A) Lineage analysis of a primary animal with P190induced CML-like disease. In the top panels, DNAs from the indicated hematopoietic tissues and lineages were digested with BglII and hybridized with a radioactive probe from the neomycin resistance gene. The blot was then stripped and reprobed with a radioactive probe from the human c-ABL gene (bottom panels), allowing determination of the proviral copy number in each sample (see Materials and Methods). LN, pooled peripheral lymph nodes; p. blood, peripheral blood; BM, bone marrow; spleen T119 ϩ , T119 Ϫ , and B220 ϩ , splenocytes purified using mAbs against TER119 and B220; per. m, purified peritoneal macrophages. Genomic DNA from a control cell line containing a single copy of the BCR/ABL provirus is on the left; DNAs from seven day 12 spleen colonies derived from bone marrow from this animal are on the right; one sample contained two distinct proviral integrants and likely represents a mixture of two individual colonies (CFU-S 12 5/6). Provirus was detected at 1.4-2.0 copies per cell in several primary tissues, indicating that one or more target cells had been transduced with multiple proviruses. Purified TER119 ϩ and B220 ϩ splenocytes contained the BCR/ABL provirus at levels equal to or higher than total spleen, indicating repopulation of erythroid and B lymphoid lineages with provirus-positive cells. (B) Lineage analysis of a primary animal with P230-induced CML-like disease. Nomenclature as in A. In this animal, provirus is absent from lymph node and thymus, but present in B220 ϩ splenocytes. Peritoneal macrophages from this animal were cultured for 1 wk in the presence of CSF-1 to increase cell number, resulting in selection for a clone containing an amplified, rearranged provirus (data not shown); because of this frequent tendency, short-term adherence was used subsequently for purification of macrophages. (C) Lineage analysis of a primary animal with P210-induced CML-like disease, and of a secondary marrow transplant recipient from this mouse that also developed CML-like disease. Nomenclature as in A. At the right, spleen and bone marrow from the secondary (2Њ) animal carry two proviruses each present at single-copy levels, indicating a doubly transduced target cell. (D) Analysis of secondary transplants from an animal with P210-induced CML-like disease. A single minor clone from the 13 clones present in the primary mouse (1Њ) was selectively recovered from a secondary animal (2Њ) with CML-like disease and from 7 out of 8 day 12 spleen colonies from this primary animal. malignancies, it was relatively easy to recognize these animals based upon clinical features and histopathology.
The survival curve of mice transplanted with P190transduced marrow differed significantly from that of P210or P230-transduced recipients (50% death in 25 d vs. 43 and 50 d, respectively; Fig. 7). Principally, this was because P190 induced B lymphoid leukemia in the majority (6/8) of recipients, which rapidly lead to the death of the host. In contrast, recipients of P210-or P230-transduced marrow that developed B lymphoid leukemia (3/9 and 5/7 mice, respectively) succumbed to these diseases much later after transplantation. The longer latency associated with P210 and P230 appeared to reflect a longer time required for establishment of the disease rather than a more aggressive leukemia, because the survival of secondary recipients transplanted with 3 ϫ 10 6 malignant lymph node cells from either P190-or P210-induced leukemia was similar, 4ف wk (data not shown). These data confirm earlier observations on the relative potency of P190 and P210 BCR/ABL for in vitro lymphoid transformation (21), and suggest that P190 induces lymphoid leukemia with shorter latency and perhaps greater efficiency than either P210 or P230.
The Lymphoid Leukemias and Macrophage Tumors Are Oligo-or Monoclonal and Lineage Restricted. To further characterize the lymphoid leukemias and macrophage tumors, we analyzed genomic DNA from the primary tumors (lymph node or pleural effusion cells for the lymphoid leukemias, and liver, ascites, or purified macrophages for the macrophage tumors). In addition, we performed lineage analysis and isolated secondary day 12 spleen colonies from selected mice. In contrast to the polyclonal nature of the CML-like syndrome, we generally observed only one or two independent proviral integrations in the lymphoid leukemias and macrophage tumors (Fig. 8, A-C). In some mice with lymphoid leukemia, different clones predominated in different anatomical sites such as lymph nodes and the pleural effusion. The presence of oligo-or monoclonal disease suggests that the presence of BCR/ABL alone is insufficient to induce these diseases, and that secondary genetic or epigenetic events are required.
In contrast to the CML-like syndrome, where provirus-positive cells were observed in multiple myeloid and lymphoid lineages in addition to the neutrophils, the provirus in the B lymphoid leukemias and macrophage tumors was confined to the tumor cells, and not present in cells of other hematopoietic lineages (Fig. 8, B and C). In animals with the macrophage disease, provirus was absent from the neutrophils, a closely related myeloid lineage. In those animals diagnosed with two diseases based on clinicopathological criteria, two different proviral integrants were usually observed, restricted to the individual tumors (26; Fig. 8 B, and data not shown). In addition, provirus was uniformly absent from day 12 spleen colonies derived from bone marrow of these mice; collectively, a total of 47 colonies derived from 3 primary animals with lymphoid leukemia and 3 primary animals with macrophage disease lacked the retroviral provirus (Fig. 8, B and C, and data not shown). These results suggest that the target cells for these diseases are progenitor cells whose differentiation capacity is lineage restricted and cannot give rise to CFU-S 12 .

Discussion
In human Ph-positive leukemia, the clear association of different forms of the BCR/ABL oncogene with distinct types of leukemia begs a biological explanation. This is most evident for P190 BCR/ABL, which is only rarely if ever observed in CML. Recently, it has been suggested that the P230 form of BCR/ABL identifies a group of patients with a distinctly benign form of CML, neutrophilic CML (14). There are two models that might explain such a phenotypic correlation with different BCR/ABL genotypes. It is possible that the three forms of BCR/ABL have different intrinsic leukemogenic activities when expressed in a hematopoietic progenitor cell that has acquired a t(9; 22) translocation. Alternatively, the three oncogenes might have identical leukemogenic properties, but their expression might be largely restricted to different hematopoietic lineages because a particular chromosome 22 breakpoint is favored in a given lineage during the formation of the Ph chromosome. For example, in this model, the BCR intron 1 breakpoint might be frequent in B lymphoid progenitor Filled symbols, CML-like disease; open symbols, B lymphoid leukemia; gray symbols, macrophage disease. Animals diagnosed with two disease processes simultaneously based on histopathological and molecular analysis are indicated by twocolor symbols. The difference in survival between recipients of P190-transduced marrow and either P210-or P230-transduced marrow was highly significant (P Ͻ 0.001, Cox test), whereas there was no significant difference between P210 and P230. BMT, bone marrow transplantation. cells but uncommon in hematopoietic stem cells, explaining the rarity of P190 BCR/ABL in CML.
Several studies of the cell of origin of the Ph chromosome translocation appear to support the latter model. In CML patients, the Ph chromosome is present in myeloid, erythroid, megakaryocytic, and B lymphoid cells, confirming that the t(9;22) translocation took place in a very early multipotential progenitor or stem cell (48). In Ph-positive ALL, about half of adult patients have traditional BCR intron 13 or 14 breakpoints on chromosome 22 generating the P210 form of BCR/ABL. Some of these patients have persistence of the Ph chromosome in remission and carry the Ph chromosome in myeloid cells and myeloid colonies grown in vitro, suggesting they represent cases of CML presenting in blast crisis after an unrecognized chronic phase (6)(7)(8)49). In contrast, the majority of ALL patients with the BCR intron 1 breakpoint characteristic of P190 BCR/ABL do not exhibit the additional cytogenetic abnormalities typical of CML blast crisis, lack the Ph chromosome in myeloid cells, and become Ph-negative during clinical remissions, suggesting they represent transformation of a cell type that is more limited in its differentiation potential (50). However, some patients with P190-associated ALL show persistence of the Ph chromosome in remission and in myeloid cells, suggesting a multipotential target cell (51)(52)(53). Detailed studies of the nature of the cell of origin in patients  Fig. 6. A single provirus is present in B lymphoid blast cells isolated from spleen (spleen B220 ϩ ) and in three independent cell lines derived from leukemic tissues, whereas three distinct proviruses are found at a total of one proviral copy per cell in malignant peritoneal macrophages. Provirus is absent from peripheral blood %08ف( neutrophils), from bone marrow and spleen cells that sedimented through Ficoll-Hypaque (ficoll pellet; entirely neutrophils and immature erythroid cells by cytospin analysis), and from splenic T lymphocytes (spleen Thy-1 ϩ ). Provirus was also absent from 14 day 12 spleen colonies derived from this mouse. (C) Lineage analysis of a mouse with BCR/ABLinduced B lymphoid leukemia. This animal demonstrated two distinct proviral integrations in tumor cells, one predominant in lymph node and the other in spleen and marrow, with both clones contributing to peripheral blood. Provirus is absent from peritoneal macrophages, bone marrow neutrophils, and decreased in thymocytes and TER119 ϩ splenocytes, both of which were contaminated with blasts at .%52ف with neutrophilic CML have not been reported. Therefore, the available clinical and molecular studies of human Ph-positive leukemia patients do not provide a biological distinction between the above two models.
The purpose of this study was to characterize the kinase activity and in vitro transformation properties of the newly described P230 Bcr/Abl protein, and to compare the in vivo leukemogenic activity of the three principal forms of Bcr/ Abl to distinguish between these alternative models. We found elevated kinase activity for P230 Bcr/Abl relative to c-Abl, although the activity of P230 was reproducibly lower than that of P210. While all three forms of BCR/ABL transformed IL-3-dependent myeloid and lymphoid cell lines to IL-3 independence, they were all less effective than IL-3 at promoting the growth of Ba/F3 lymphoid cells, with the rates of proliferation directly correlating with the intrinsic tyrosine kinase activity of Bcr/Abl. The reason for this difference is not known, but it is plausible that increased tyrosine phosphorylation of critical substrates such as STAT6 by P190 might contribute to proliferation.
To test the hypothesis that the three forms of BCR/ABL have different leukemogenic properties in vivo, we used the bone marrow transduction/transplantation model system. This assay allowed us to directly determine whether P190 is able to cause a CML-like syndrome and whether P230 is less potent than P210 upon transduction of an identical spectrum of primary hematopoietic cells. When the three forms of BCR/ABL were transduced into marrow from donors treated with 5-FU, they induced an identical fatal CML-like disease in recipients transplanted with the transduced cells, with no appreciable differences in histopathology, hematologic parameters, or disease latency. These observations argue that the three forms of BCR/ABL induce an identical proliferative stimulus to myeloid cells in our experimental model. Human patients with neutrophilic CML are described as having lower peripheral blood leukocyte counts, less splenomegaly, and requiring little or no myelosuppressive therapy (14), features which imply less myeloid cell proliferation and expansion relative to traditional CML. However, our results do not support the hypothesis that P230 generates a weaker proliferative stimulus in myeloid cells than P210, and suggest that the mild clinical symptoms observed in some patients with P230 BCR/ABL are due to other variables, such as genetic differences between individuals or a selection bias (15)(16)(17). In recipients of transduced marrow from non-5-FU-treated donors, there is a suggestion of increased latency of P210-and P230-induced CMLlike disease compared with P190, which leaves open the possibility of differences in BCR/ABL-induced CML-like disease under conditions where small numbers of proviral clones and perhaps lower Bcr/Abl expression exist.
Similarly, because P190 BCR/ABL can induce a CMLlike syndrome in mice which is identical to that induced by P210, it is difficult to argue that the rarity of P190 in human CML reflects different leukemogenic activity of P190 in myeloid cells. Rather, our results suggest that the restriction of P190 BCR/ABL to human acute leukemias may be due to a relative lack of BCR first intron breakpoints dur-ing the generation of the Ph chromosome in stem cells. Alternatively, patients with the P190 form of BCR/ABL may develop the Ph translocation in a stem cell but manifest only a brief chronic phase. This is supported by a recent study of human P190-positive ALL, where the Ph chromosome was detected by interphase fluorescence in situ hybridization in granulocytes of five out of five patients, suggesting multilineage involvement (53).
Although there was no difference between the three BCR/ABL oncogenes in induction of a CML-like disease in recipients of transduced marrow from 5-FU-treated donors, there was a highly significant difference in survival of recipients of P190-transduced marrow compared with recipients of P210-and P230-transduced marrow when donors were not pretreated with 5-FU. Under these conditions, rather than succumbing to CML-like disease, most recipients of P190-transduced marrow developed B lymphoid leukemia. Although several recipients of P210-and P230-transduced marrow also developed B lymphoid leukemia, the disease developed significantly later with P210 and P230. These results demonstrate that P190 BCR/ABL induces lymphoid leukemia in vivo with shorter latency than P210 or P230, and are in agreement with previous studies which found P190 to be more potent than P210 at transformation of B lymphoid cells in vitro (21) and induction of lymphoid leukemia after marrow transduction (32). Our data further suggest that P230 and P210 have similar lymphoid leukemogenic activity. The mechanism of the increased lymphoid leukemogenic activity of P190 relative to P210 and P230 is not known. One possibility is that the increased intrinsic tyrosine kinase activity of P190 allows elevated tyrosine phosphorylation of a substrate critical to proliferation or transformation of lymphoid cells. One candidate for such a key substrate is STAT6, whose DNAbinding activity is activated via tyrosine phosphorylation by P190 but not P210 (24). In support of this, we have observed that bone marrow from stat6 Ϫ/Ϫ mice (23) exhibits significant resistance to transformation by P190 BCR/ABL in vitro when compared with stat6 ϩ/ϩ marrow (our unpublished observations). An alternative possibility is that the presence of distinct functional motifs in the Bcr portion of P210 and P230 BCR/ABL impairs lymphoid transformation by these oncogenes. The only known domain common to P210 and P230 but lacking in P190 is a homology to Cdc24Hs/Dbl, which is a member of a class of guanine nucleotide exchange factors for small G proteins of the Rac/Cdc42 family. Our system allows us to readily test the importance of this domain in lymphoid leukemogenesis.
What accounts for the very different spectrum of leukemia observed when non-5-FU-treated marrow is used? Data in this report argue that the CML-like disease, B lymphoid leukemia, and macrophage disease are the consequence of BCR/ABL transduction of distinct bone marrow target cells, and the relative abundance and/or infectability of these target cells is influenced by 5-FU treatment of donors. In mice with the CML-like syndrome, multiple myeloid and lymphoid lineages carry the same spectrum of proviral clones, demonstrating that the target cell is a pro-genitor with both lymphoid and myeloerythroid differentiation potential. In addition, we found the BCR/ABL provirus in the majority of secondary day 12 spleen colonies derived from primary mice with the CML-like syndrome, demonstrating that some of these target cells can generate CFU-S 12 . In contrast, the bone marrow target cells for induction of BCR/ABL-induced B lymphoid leukemia and macrophage disease have restricted in vivo differentiation potential, so that provirus-positive cells do not contribute to other hematopoietic lineages; further, the provirus is never found in day 12 spleen colonies derived by secondary transplantation of marrow from these animals. These observations suggest the BCR/ABL B lymphoid leukemia target cell is a committed B lymphoid progenitor similar or identical to the Abelson virus target cell, which is known to be an immature Thy-1 lo B220 ϩ committed lymphoid progenitor (54) that is distinct from CFU-S 12 (55).
Analysis of proviral integration patterns suggests other pathogenetic differences between BCR/ABL-induced CMLlike disease and B lymphoid leukemia. The CML-like disease observed in recipients of transduced marrow from 5-FU-treated donors is polyclonal, whereas the B lymphoid leukemias are mono-or oligoclonal. The short latency of the CML-like disease together with the polyclonal nature suggest that BCR/ABL alone is capable of inducing CMLlike disease, whereas the longer latency and monoclonal nature of the B lymphoid leukemias imply that expression of BCR/ABL alone may be insufficient for malignant transformation of primary B lymphocytes and that additional events are required, a concept supported by many previous observations (20,(56)(57)(58)(59). However, proof of the sufficiency of BCR/ABL for induction of the CML-like disease will require limiting dilution analysis and knowledge of the efficiency of infection of the CML target cell.
Interestingly, we and others (29) have found evidence for heterogeneity among the presumptive target cells for the murine CML-like syndrome. Of the many clones contributing to the expansion of myeloid cells in several primary CML animals, only one or two relatively minor clones were observed to contribute to day 12 spleen colonies and to efficiently transfer the CML-like disease in secondary transplants. These data imply two distinct target cells for the murine CML-like syndrome, both with the capacity for multilineage differentiation in the primary animal, but with different ability for repopulation of secondary recipients. These properties are reminiscent of the hierarchy of murine hematopoietic stem cells, where a distinct population of progenitor cells enriched for CFU-S 12 has transient multilineage repopulating and self-renewal potential, and a more differentiated subset lacks secondary transplantability (60). An alternative possibility is that all the CML target cells are equivalent immediately after infection, but additional genetic or epigenetic abnormalities confer transplantability on a small subset of transduced progenitors. Distinguishing between these models will require isolation of the various target cells from whole marrow.
We found small to medium-sized accumulations of macrophages in the livers of nearly every animal with the CMLlike syndrome. Previous studies have shown that some animals with extensive BCR/ABL-induced macrophage tumors exhibit elevated serum GM-CSF and/or G-CSF levels, and a secondary elevation in peripheral blood neutrophils that lack the retroviral provirus and are therefore not a direct part of a malignant process (26,30,44). In our study, immunoassay of plasma from several mice with primary or secondary CML-like disease revealed normal levels of GM-CSF but increased levels of IL-3 (data not shown). The biological significance of the elevation in plasma IL-3 in these animals is not clear and is under investigation. However, in contrast to mice with primary macrophage tumors, the neutrophils in all animals with the CML-like disease contain the retroviral provirus and express Bcr/Abl protein, demonstrating that this disease represents a bona fide myeloproliferative process. The lineage analysis suggests that the macrophage collections in mice with the CML-like syndrome represent the product of differentiation of the myeloid clones, as previously suggested (32).
Our original goal in expressing BCR/ABL in the murine hematopoietic system by retroviral transduction (26) was to determine whether BCR/ABL was the direct cause of CML, a point which has been established beyond reasonable doubt (26)(27)(28)(29). However, our initial model system was difficult to use for comparative studies because of the low efficiency and variability of induction of the CML-like disease. In this report, the mouse bone marrow transduction/transplantation system has been improved, and we have used this system for a direct comparison of the leukemogenic properties of the three principal forms of BCR/ABL. We found no significant difference in the ability of the three oncogenes to induce a CML-like myeloproliferative syndrome, but observed that P190 BCR/ ABL had increased potency for induction of B lymphoid leukemia. This model system should be very useful for studying the molecular pathophysiology of the Ph-positive leukemias, such as testing the requirement for various functional domains of Bcr/Abl, determining signaling pathways relevant to leukemogenesis, and investigating the physiological effects of BCR/ ABL expression in primary hematopoietic cells.


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cDNA Cloning of a Novel Androgen Receptor Subtype*

There has been general acceptance that only one type of androgen receptor (AR) exists in an individual. This contrasts with other members of the nuclear receptor superfamily where multiple forms have been reported (e.g. estrogen receptor α/β, thyroid hormone receptor α/β, etc.). We have previously identified 11-ketotestosterone (a potent androgen in teleosts) as the spermatogenesis-inducing hormone of the Japanese eel and have cloned its receptor (eAR1) cDNA from eel testis. Here we report on the cloning of a cDNA encoding a second type of AR (eAR2) from the eel testis and the functional characterization of the encoded protein. This cDNA contains a complete open reading frame encoding 797 amino acid residues. The amino acid sequence of eAR2 shows high homology with other ARs, including eAR1, in the DNA-binding (98–88%) and ligand-binding (59–85%) domains, whereas the other domains show low homology (<35%). In transient transfection assays of mammalian cells, the eAR2 protein displayed androgen-dependent activation of transcription from the androgen-responsive murine mammary tumor virus promoter. Tissue distribution of its mRNA was different from that of eAR1. We conclude that eAR2 is a novel AR in the eel, which we suggest should be named eel ARβ to distinguish it from eAR1 (eARα).

Since the initial cDNA cloning of nuclear steroid hormone receptors, the superfamily of nuclear receptors has rapidly grown. Nuclear receptors possess highly conserved DNA-binding domains and moderately conserved ligand-binding domains (LBDs) 1 (1). It has been shown that members of this superfamily, such as estrogen receptor (ER), retinoic acid receptor, retinoid X receptor, and thyroid hormone receptor, have multiple subtypes and isoforms (1,2). In the case of the ER, two subtypes (ER␣ and ER␤) exist, and ER␤ has several isoforms designated ER␤1-5 (3) or ER␤cx (4). Two isoforms of cDNA encoding rainbow trout AR (designated AR␣ and AR␤) (5) have been reported and are probably derived from salmonid tetraploidy. A novel subtype of the eel AR may exist because a cDNA encoding part of another AR was isolated during cloning of eel AR (eAR1) from testis (6).
In vertebrates, differentiation of the masculine phenotype is directed by secretion of steroid hormones, usually androgen (7). Recent studies in our laboratory, using a teleost, the Japanese eel (Anguilla japonica), have identified 11-ketotestosterone (11KT), a major androgen in some fish, as the spermatogenesisinducing hormone (8 -10). 11KT can induce the entire process of spermatogenesis from spermatogonial proliferation to sperm formation in both cultured testicular fragments (9) and cocultured germ and somatic cells (10) from immature eels. 11KT activates Sertoli cells to stimulate production of activin B, which has been identified as the spermatogonial proliferator in the Japanese eel (11). However, the mechanisms underlying the regulation of eel spermatogenesis by 11KT are not fully understood. To understand the mechanism of androgen action and AR regulation of gene transcription, it is important to isolate and characterize subtypes of the AR.
Here, we describe the isolation and characterization of an eel cDNA containing an entire AR subtype coding region. The transactivation function of this AR was determined by expressing the cDNA in transiently transfected human kidney 293 cells. The AR mRNA levels in various tissues from eels were measured by reverse transcription-polymerase chain reaction (PCR).

EXPERIMENTAL PROCEDURES
Animals and Treatment-Cultivated Japanese eel males (150 -200 g in body weight) were purchased from a commercial eel supplier. They were kept in recirculating freshwater tanks with a capacity of 500 liters at 20°C. Fish were not fed throughout the experimental period.
Isolation of cDNA Clones-A cDNA fragment of an AR subtype (eAR2), whose sequence partially differed from eAR1, was isolated after a testis cDNA library from Japanese eels killed 1 and 3 days after human chorionic gonadotropin injection was screened using a PCR amplified product as a probe (6). Because the 3Ј terminus of the clone was truncated, 3Ј-rapid amplification of cDNA ends (3Ј-RACE) was performed using a Marathon cDNA amplification kit (CLONTECH). The insert cDNA was nest-deleted at both ends using ExoIII/Mung bean nuclease. Sequencing was performed using the ABI PRISM Dye Terminator cycle sequencing kit (Applied Biosystems).
Construction of Plasmid Vectors-pcDNA-eAR2 was constructed by PCR amplification of the entire protein coding region (amino acids 1-798) of the eAR2, using primers that introduced an EcoRI site and in-frame Kozak sequence (12) at the 5Ј end and an EcoRI site at the 3Ј end. The EcoRI fragment was inserted at the EcoRI site of pcDNA3.1(ϩ) (Invitrogen). pcDNA-eARrev, which had the pcDNA-eAR2 inserted in the reverse direction, was also prepared as a negative control. pcDNA-eAR1 has been described elsewhere as pcDNA-eAR (6). An androgen-regulated reporter vector, named pGV2-MMTV, was also constructed as described previously (6).
Transactivation Assays-Human embryonic kidney 293 cells were seeded in 24-well plates at 5 ϫ 10 4 cells/well in phenol-red free Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% charcoalstripped fetal calf serum, and 50 g/ml kanamycin. After 24 h, the cells were transfected with 250 ng of pGV2-MMTV, 250 ng of pRL-TK (for internal control to normalize for variation in transfection efficiency; contains the Renilla reniformis luciferase gene with the herpes simplex * This work was supported in part by Grants-in-aid for Research for the Future (JSPS-RFTF 96L00401) and Priority Areas (07283104) from the Japanese Ministry of Education, Science, Sports, and Culture, Japan, and Bio Design Program from the Ministry of Agriculture, Forestry and Fisheries, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB025361.
virus thymidine kinase promoter; Toyo Ink), and 50 ng of pcDNA-eAR2, pcDNA-eAR1, or pcDNA-eARrev, using Tfx TM -20 reagent (Promega) according to the manufacturer's instructions. After 1 h of incubation, 1 ml of fresh medium containing 10% fetal calf serum plus steroid hormones was applied to the cells. After 48 h, the cells were collected, and the luciferase activities of the cells were measured by a chemilumines- cence assay using PicaGene Dual TM SeaPansy Assay System kit (Promega) according to the manufacturer's instructions.
Reverse Transcription-PCR-Poly(A) ϩ RNA was prepared from gill, heart, head kidney, spleen, liver, muscle, testis, and brain of male eels and ovary of female eels by a method described previously (13). Random primed double strand cDNA was synthesized using a cDNA synthesis system (Amersham Pharmacia Biotech).
Statistics-Data from transactivation experiments were analyzed by one-way analysis of variance. Significant differences between groups were identified by the Fisher's protected least significant difference test. Differences were regarded as significant at p Ͻ 0.01.

RESULTS AND DISCUSSION
Sequence Homology with Other Steroid Hormone Receptors-During cloning of eAR1 cDNA, one positive clone, whose sequence partially differed from that of eAR1 cDNA, was ob-tained. This insert is 1,718 base pairs in size, and the 3Јterminus of the cDNA was truncated. Thus, 3Ј-rapid amplification of cDNA ends (3Ј-RACE) was performed by using primer (corresponding to 1,594 -1,622 base pairs) designed so that the PCR product overlapped with the 3Ј terminus truncated clone at an eAR2 unique sequence. Based on the 3Ј terminus truncated clone and the 3Ј-RACE product obtained, the nucleotide sequence and the deduced amino acid sequence of eAR2 were determined (Fig. 1). The sequence contains a long open reading frame encoding 797 amino acid residues (molecular mass, 94,688 Da). Comparison of the amino acid sequence of eAR2 with those of eel steroid hormone receptors and ARs of several species is shown in Fig. 2. The eAR2 gene sequence could be subdivided into 4 -5 domains (A-F) as defined by Krust et al. (15). The putative DNA-binding domain (residues 436 -522) and LBD (residues 548 -797) show high homology with those of other ARs including eAR1 (DNA-binding domain, 98 -88%; LBD, 59 -85%). The other domains show low homology (Ͻ35%). Judging from these structures, eAR1 and eAR2 are not isoforms derived from translational initiation at two in-phase ATG codons, alternative splicing, or tetraploidy.
In tetraploid salmonids, two sets of genes for certain proteins have been found (16 -18). Recently, two isoforms of AR (designated rtAR␣ and rtAR␤) were reported in rainbow trout (5). They are probably derived from salmonid tetraploidy, because homology of their full-length open reading frames is very high (85% in amino acid residues) compared with that of eAR1 and eAR2 (40% in amino acid residues). Notably, rtAR␤ did not activate transcription of an androgen-responsive gene. It has been reported that two types of gonadotropin ␣-subunit exist not only in salmonids but also in tetraploid common carp (19). Interestingly, when these subunits of common carp were expressed in insect cells by recombinant baculovirus, ␣1 associated with ␤ subunit to form active gonadotropin, whereas ␣2 associated with ␤ subunit but the resulting entity did not display gonadotropic activity (19). Thus, rtARs appear to be isoforms derived from tetraploidy as well as gonadotropin ␣ of carp. Moreover, both rtARs are similar to eAR2 and different from eAR1 in structure. On the other hand, tetrapod ARs are similar to eAR1. Whether a subtype corresponding to eAR2 exists in tetrapods as well as other teleosts is an interesting question.
Although the N-terminal A/B domain displayed low homology overall, one conserved region is found among the ARs (residues 117-135 in the eAR2). It has been shown that the A/B domain contains transactivation function, which is dependent on cell and promoter types but less on ligand binding (20). Thus, it is interesting to determine whether the conserved regions in the A/B domain of ARs are involved in common functions, such as cell/promoter-specific transactivation.
In the DNA-binding domain, the position of cysteine residues, which constitute the two zinc finger motifs, and the P box (GSCKV), which is the important region for determination of the target gene (21), are also conserved in the eel AR2. Several amino acids in the LBD, which have been predicted as important in ligand binding based on crystallographic, genetic, or biochemical analysis (22), differ between hAR and eel ARs.  graphically. a, b, c, d, e, w, x, y, z, groups sharing the same letter codes were not significantly different at p Ͻ 0.01, and groups from a to e and from w to z were significantly different from vehicle-group of eAR2 and eAR1, respectively, at p Ͻ 0.01. Poly(A) ϩ RNA was prepared from gill, heart, head kidney, spleen, liver, muscle, testis, and brain of male eels and ovary of female eels. ␤-actin was used as a positive control. Messenger RNA of both AR subtypes were detected by reverse transcription-PCR (see "Experimental Procedures"). eARs for various androgens may not differ greatly from that of the hAR. In addition, a leucine zipper motif, which was found in the C-terminal region of eAR1 LBD (residues 787-815) (6), is also conserved in the corresponding region of eAR2 (residues 736 -764). The leucine zipper-like structure is conserved in many nuclear receptors, and is required for dimerization of receptors (23). Thus, this region in the eel AR could be involved in dimerization of ARs.
There were three minor differences in ligand affinity between eAR2 and eAR1, although the difference in homology of LBD between eAR1 and eAR2 (77%) is smaller than that between rat ER␣ and ER␤ (53.5%) (2). First, synthetic androgens were significantly more effective than 5␣DHT in inducing transcriptional activity of eAR2, a finding that differs from the pattern seen for eAR1 (Fig. 3B). Second, androstenedione induced weak transcriptional activity in eAR2, whereas eAR1 was not significantly stimulated by this steroid. Third, 17␣,20␤-dihydroxy-4-pregnen-3-one, which is the maturationinducing steroid of many teleosts, did not induce any significant transcriptional activity of eAR2, whereas it induced weak transcriptional activity of eAR1. The ligand binding affinity of rat ER␣ and ER␤ protein for physiological ligands is overall quite similar, certainly when only the order of competition is compared (24). It was also reported that the affinity for other estrogen-related substances (moxestrol, 17␣-estradiol, etc.) differed between both ER subtypes (24). Similar differences between eel ARs were also observed in this study.
What is the native ligand of eARs? In the Japanese eel, 11KT (and not testosterone or 5␣DHT) is the major androgen (quantitatively and physiologically) produced by testis during spermatogenesis (8,9). 11KT stimulated luciferase activity through eAR2 and eAR1 (6) in a dose-dependent manner (Fig. 4). The minimum stimulatory dose of 11KT was 1 nM. Serum 11KT levels in human chorionic gonadotropin-treated and untreated Japanese eel males ranged from 0.8 to 26 nM (8). Physiological doses of 11KT are thus sufficient to activate luciferase activity via eel ARs. Thus, these results indicate that the major native ligand of ARs is 11KT.
Tissue Distribution of mRNA-The levels of AR mRNA in various tissues from male Japanese eel were measured by reverse transcription-PCR. eAR2 mRNA was detected only in spleen, muscle, and testis, whereas eAR1 mRNA was observed in several other tissues as well (Fig. 5). Tissue distribution and/or the relative level of ER␣ and ER␤ mRNA expression is quite different in rat (24). Differences in tissue distribution mRNA for eel ARs are predictable because their A/B domains, which contain a region of transactivation function strongly dependent on cell and promoter types but less on ligand binding, show low homology. Thus, difference in transactivation function of ARs on certain target genes may also exist in eel.
These results indicate that eAR2 is a novel AR subtype. Therefore, we suggest that this AR subtype be named eel AR␤ to differentiate it from eAR1 (AR␣) cloned previously (6). The biological significance of the existence of two different ARs is unclear and is being addressed in ongoing studies.