Search

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-beta3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-beta3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-beta3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.

The Philadelphia (Ph) chromosome is found in approximately 3% of pediatric patients with acute lymphoblastic leukemia (ALL) and the percentage markedly increases in adult patients. The prognosis for this class of patients is poor, and no standard chemotherapy combination so far has demonstrated long-term efficacy. The Ph-translocation joins the BCR and ABL genes and leads to expression of a chimeric Bcr/Abl protein with enhanced tyrosine kinase activity. This increase in activity leads to malignant transformation by interference with basic cellular functions such as the control of proliferation, adherence to stroma and extracellular matrix, and apoptosis. One important pathway activated by Bcr/Abl is the Ras pathway. Ras proteins have to undergo a series of posttranslational modifications to become biologically active. The first modification is the farnesylation of the C-terminus catalyzed by farnesyl transferase. We studied the effect of the farnesyl transferase inhibitor SCH66336 in an in vivo murine model of Bcr/Abl-positive acute lymphoblastic leukemia. In the early leukemic phase, mice were randomly assigned to a treatment, a vehicle, and a nontreatment group. The treatment was well tolerated without any detectable side effects. All animals of the control groups died of leukemia/lymphoma within 103 days (range, 18-103 days). In contrast, 80% of the drug-receiving group survived without any signs of leukemia or lymphoma until termination of treatment, after a median treatment period of 200 days (range, 179-232 days). We conclude that farnesyl transferase inhibitor SCH66336 is able to revert early signs of leukemia and significantly prolongs survival in a murine ALL model.

The adapter protein Crkl has been implicated in the abnormal signal transduction pathways activated by the Bcr/Abl oncoprotein, which causes Philadelphia-positive leukemias in humans. To investigate the role of Crkl in tumorigenesis, we have generated transgenic mice that express human Crkl from the CRKL promoter. Western blot analysis showed a 4-6-fold overexpression of transgenic Crkl above endogenous crkl in two lines and increased constitutive complex formation between Crkl and C3G, an exchange factor for the small GTPase Rap1. This was associated with a significant increase in integrin-based motility of transgenic macrophages. Overexpression of Crkl was associated with increased incidence of tumor formation, and Rap1 was activated in a metastatic mammary carcinoma. The coexpression of Crkl and Bcr/Abl in mice transgenic for P190 BCR/ABL and CRKL markedly increased the rapidity of development of leukemia/lymphoma, decreasing the average survival by 3.8 months. These results provide direct evidence that Crkl plays a role in tumor development and is important in the leukemogenesis caused by Bcr/Abl.

The deregulated Bcr/Abl tyrosine kinase is responsible for the development of Philadelphia (Ph)-positive leukemia in humans. To investigate the significance of the C-terminal Abl actin-binding domain within Bcr/Abl p190 in the development of leukemia/lymphoma in vivo, mutant p190 DNA constructs were used to generate transgenic mice. Eight founder and progeny mice of 5 different lines were monitored for leukemogenesis. Latency was markedly increased and occurrence decreased in the p190 del C lines as compared with nonmutated p190 BCR/ABL transgenics. Western blot analysis of involved hematologic tissues of the p190 del C transgenics with end-stage disease showed high-level expression of the transgene and tyrosine phosphorylation of Cbl and Hef1/Cas, proteins previously shown to be affected by Bcr/Abl. These results show that the actin-binding domain of Abl enhances leukemia development but does not appear to be an absolute requirement for leukemogenesis.

The activated tyrosine kinase, Bcr-abl, is implicated in a number of hematopoietic malignancies. The exact biological mechanism by which the kinases transforms cells is still not well delineated. Previous data has suggested that the inhibition of apoptosis and the deregulation of cell cycle progression as the result of P210Bcr-abl expression might contribute to leukemogenesis. In vitro systems in which Bcr-Abl is over-expressed have concluded that similar growth regulatory pathways are affected as a result of the expression of both P210 and P190Bcr-abl. Here, we utilized an in vitro P190Bcr-abl leukemia mouse model to dissect the early events that contribute to transformation by this isoform of Bcr-Abl. In this mouse model P190Bcr-abl is expressed as a low but physiologically relevant level in that all mice develop pre-B leukemia lymphomas. We show that cell cycle and apoptotic responses to DNA damage are intact in bone marrow and spleen cells of such animals. We also demonstrate a normal induction of p21WAF-1/CIP1 in both hematopoietic and non-hematopoietic tissue as a result of genotoxic stress. We suggest that P190Bcr-abl induced transformation is different than that of P210Bcr-abl.

We used a sensitive, quantitative bisulfite PCR assay, methylation sensitive single nucleotide primer extension (Ms-SNuPE), to measure methylation of the 5' CpG islands of c-abl and p15 in chronic myelogenous leukemia (CML) patients during progression. We found that the Pa promoter of c-abl was methylated in 81% (17/21) of the white blood cells (WBCs) of CML patients, which correlates with previous reports. In contrast, WBCs from healthy donors, acute myelogenous leukemias, acute lymphocytic leukemias, and myelodysplastic syndromes were unmethylated at the c-abl Pa promoter locus. We also observed p15 hypermethylation in 24% (8/34) of CML cases. Methylation of the p15 but not c-abl Pa promoters was associated with CML progression (P = 0.047 vs 0.46), and the two events were independently acquired. We conclude that de novo methylation of c-abl and p15 both occur in CML, and analysis of DNA methylation changes using the bisulfite-based MS-SNuPE assay allows both a sensitive and quantitative assessment of these molecular events compared to other methods currently utilized. (Blood. 2000;95:2990-2992)

After cessation of lactation, the mammary gland undergoes involution, which is characterized by a massive epithelial cell death and proteolytic degradation of the extracellular matrix. Whereas the expression patterns and also the function of TGF-beta isoforms during mammary gland branching morphogenesis and lactation are well understood, their expression during postlactational involution and therefore a possible role in this process is poorly known. In this study we show that TGF-beta3 expression is dramatically induced (>fivefold) during mouse mammary gland involution when compared to that of virgin mouse, reaching a maximal expression level at day 4 after weaning. In contrast, other TGF-beta isoforms do not display significant increase in expression during involution (TGF-beta1, 1.3-fold and TGF-beta2, <1.5-fold) when compared to that of virgin or lactating mice. During mammary gland involution, TGF-beta3 is expressed in the epithelial layer and particularly in myoepithelial cells. A comparison of the kinetics of TGF-beta3 expression to that of programmed cell death and degradation of the basement membrane suggests that TGF-beta3 functions in the remodeling events of the extracellular matrix during the second stage of involution.

Aspartylglycosaminuria (AGU), the most common lysosomal disorder of glycoprotein degradation, is caused by deficient activity of glycosylasparaginase (AGA). AGA-deficient mice share most of the clinical, biochemical and histopathologic characteristics of human AGU disease. In the current study, recombinant human AGA administered i.v. to adult AGU mice disappeared from the systemic circulation of the animals in two phases predominantly into non-neuronal tissues, which were rapidly cleared from storage compound aspartylglucosamine. Even a single AGA injection reduced the amount of aspartylglucosamine in the liver and spleen of AGU mice by 90% and 80%, respectively. Quantitative biochemical analyses along with histological and immunohistochemical studies demonstrated that the pathophysiologic characteristics of AGU were effectively corrected in non-neuronal tissues of AGU mice during 2 wk of AGA therapy. At the same time, AGA activity increased to 10% of that in normal brain tissue and the accumulation of aspartylglucosamine was reduced by 20% in total brain of the treated animals. Immunohistochemical studies suggested that the corrective enzyme was widely distributed within the brain tissue. These findings suggest that AGU may be correctable by enzyme therapy.-Dunder, U., Kaartinen, V., Valtonen, P., Väänänen, E., Kosma, V.-M., Heisterkamp, N., Groffen, J., Mononen, I. Enzyme replacement therapy in a mouse model of aspartylglycosaminuria.

Newborn transforming growth factor (TGF)-beta3-null mutant mice exhibit defects of palatogenesis and pulmonary development. Glucocorticoids, which play a central role in fetal lung maturation, have been postulated to mediate their stimulatory effects on tropoelastin mRNA expression through TGF-beta3 in cultured lung fibroblasts. In the present study, we analyzed the abnormally developed lungs in TGF-beta3-null mutant mice and compared the effects of glucocorticoids on gene expression and lung morphology between TGF-beta3 knockout and wild-type mice. Lungs of TGF-beta3-null mutant mice on embryonic day 18.5 did not form normal saccular structures and had a thick mesenchyme between terminal air spaces. Moreover, the number of surfactant protein C-positive cells was decreased in TGF-beta3-null mutant lungs. Interestingly, glucocorticoids were able to promote lung maturation and increased expression of both tropoelastin and fibronectin but decreased the relative number of surfactant protein C-positive cells in fetal lungs of both genotypes. This finding provides direct evidence that glucocorticoid signaling in the lung can use alternative pathways and can exert its effect without the presence of TGF-beta3.

The SH2-SH3 adaptor protein Crkl has been implicated in the signal transduction pathways of several membrane-bound receptors. Tyrosine phosphorylation of proteins associated with such signalling complexes can generate binding sites for the Crkl SH2-domain and can recruit proteins constitutively bound to Crkl via the Crkl SH3 domain into such complexes. In the current study we show that Crkl, but only a minor amount of the related Crk, form constitutive complexes in vivo with guanine nucleotide exchange factor C3G in 3T3 fibroblasts. Adhesion of both normal and transformed cells to fibronectin or other extracellular matrix proteins consistently induces the tyrosine-phosphorylation of C3G. Adhesion-induced tyrosine phosphorylation of C3G is dependent on an intact cytoskeleton and peaks at 5-10 min after attachment. In contrast, 3T3 cells stably transfected with Bcr/Abl P210 show a prominent reduction in the amount of C3G complexed to Crkl and do not exhibit tyrosine-phosphorylation of C3G upon spreading and attachment. These data establish that integrin-mediated cell adhesion results in Crkl-mediated tyrosine phosphorylation of C3G, a pathway which can be disrupted by Bcr/Abl.

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.

The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120(cas) and elevated tyrosine phosphorylation of p120(cas). beta-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and beta-catenin were found to be in complex. Significantly, although the levels of alpha-catenin, beta-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of alpha-catenin and beta-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion.

Aspartylglycosaminuria (AGU) is one of the most common lysosomal storage disorders in humans. A mouse model for AGU has been recently generated through targeted disruption of the glycosylasparaginase gene, and at a young age the glycosyl asparaginase-deficient mice demonstrated many pathological changes found in human AGU patients (Kaartinen V, Mononen I, Voncken J-W, Gonzalez-Gomez I, Heisterkamp N, Groffen J: A mouse model for aspartylglycosaminuria. Nat Med 1996, 2:1375-1378). Our current findings demonstrate that after the age of 10 months, the general condition of null mutant mice gradually deteriorated. They suffered from a progressive motoric impairment and impaired bladder function and died prematurely. A widespread lysosomal hypertrophy in the central nervous system was detected. This neuronal vacuolation was particularly severe in the lateral thalamic nuclei, medullary reticular nuclei, vestibular nuclei, inferior olivary complex, and deep cerebellar nuclei. The oldest animals (20 months old) displayed a clear neuronal loss and gliosis, particularly in those regions, where the most severe vacuolation was found. The severe ataxic gait of the older mice was likely due to the dramatic loss of Purkinje cells, intensive astrogliosis and vacuolation of neurons in the deep cerebellar nuclei, and the severe vacuolation of the cells in vestibular and cochlear nuclei. The impaired bladder function and subsequent hydronephrosis were secondary to involvement of the central nervous system. These findings demonstrate that the glycosylasparaginase-deficient mice share many neuropathological features with human AGU patients, providing a suitable animal model to test therapeutic strategies in the treatment of the central nervous system effects in AGU.

The BCR gene contributes to Philadelphia-positive leukemogenesis via a number of discrete mechanisms, one of which may be through interaction of its normal gene product with the Bcr/Abl oncoprotein. In the current study this hypothesis was tested in vivo by introducing a Bcr/Abl P190 transgene into mice lacking endogenous bcr protein. Our finding, that the P190 BCR/ABL oncogene is still capable of producing leukemia in these mice with indistinguishable latency and clinical pattern as in genetically matched counterparts, rules out any significant or major contribution of the bcr protein as a whole to leukemia development in these mice.

BCR/ABL is considered responsible for the development of Philadelphia chromosome-positive leukemia. Experimental animal models, such as transgenic mice, have demonstrated unambiguously that Bcr/Abl is capable of inducing leukemogenesis. The adaptor molecule Crkl is a major in vivo substrate of the deregulated Bcr/Abl tyrosine kinase and functions as a molecular link with other signaling proteins. While associated in vivo with Bcr/Abl through its SH3 domain, Crkl can interact simultaneously via its SH2 domain with other tyrosine-phosphorylated proteins. Here we report the identification of prominently tyrosine-phosphorylated proteins with a molecular mass of approximately 110 kDa, which bind specifically to the Crkl SH2 domain in leukemic tissues of P190BCR/ABL transgenic mice. We demonstrate that these proteins are identical to Hef1/Cas-L, which is related to p130(Cas). The proto-oncoprotein p120(Cbl) and Hef1, but not p130(Cas), were detectably phosphorylated on tyrosine in P190Bcr/Abl-expressing leukemic cells and were found in complex with Crkl, showing the existence of protein complexes in P190Bcr/Abl leukemic cells, consisting of P190Bcr/Abl, Crkl, and Hef1 or p120(Cbl). This supports a model in which Crkl acts as mediator between Bcr/Abl and downstream effectors. Since Hef1 is involved in the beta1-integrin signaling pathway, our study demonstrates that Bcr/Abl could specifically interfere with normal beta1-integrin signaling.

The small GTP-binding proteins Rac1 and Rac2 are critically important in regulating multiple signal transduction pathways in eukaryotic cells. Here we report the isolation of a novel third Rac family member, Rac3. Rac3 differs from Rac1/2 at its carboxyl-terminal end, a domain associated with subcellular localization and binding to specific cellular regulators. RAC3 mRNA expression patterns differ from those of RAC2, which is hematopoietic specific and also from those of RAC1. The RAC3 gene was mapped to chromosome 17q23-25, a region frequently deleted in breast cancer. Rac3 protein levels are not affected by organization of the actin cytoskeleton but remarkably, are serum-inducible. Rac3 is an active GTPase, and this activity is regulated by Bcr. When constitutively activated, Rac3 is able to stimulate efficiently the c-Jun amino-terminal kinase signaling pathway. These findings support a role for Rac3 in intracellular signaling.

Studies on transforming growth factor beta3 (TGF-beta3) deficient mice have shown that TGF-beta3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF-beta3 in palatogenesis, we cultured TGF-beta3 null mutant and wild-type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF-beta3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF-beta3 (-/-) samples is not impaired adhesion. Our data define a specific role for TGF-beta3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane.

The 1982 discovery that in chronic myeloid leukaemia (CML) the ABL proto-oncogene is translocated to the BCR gene located on chromosome 22 initiated many studies on the structural organization and function of these genes. The nucleotide sequence of the entire BCR and major parts of the ABL gene has now been determined. However, the actual cause of the fusion of BCR with ABL remains essentially unknown. Mouse models have been helpful to unravel the normal cellular function of BCR and ABL, as well the activity of BCR-ABL, although a single mechanism explaining the transforming activity of the latter has not been discovered. The cause of progression of the disease remains unknown, and no single genetic abnormality has been linked to the blast phase of CML. Much has been learned concerning the molecular biology of CML, but answers to the fundamental questions above may be expected in the coming years in parallel to increasing knowledge of genome structure, signal transduction and cell cycle control.

The fusion of the mandibular prominences along the midline is achieved with the absence of medial epithelial cells at the fusion site. Failure of fusion of the mandibular prominences results in median cleft of the lower lip and mandible. Cellular and molecular events controlling mandibular fusion were examined during the fusion process in mouse embryogenesis. Cell lineage analyses at the fusion site revealed that epithelial cells migrated to the surface and oral epithelia. DiI-labeled epithelial cells were not observed within the mandibular mesenchyme at any state of fusion. Examination of the midline region did not reveal cells with ultrastructural changes characteristic of apoptotic cell death. An increase in lysosomal enzymes in the midline epithelial cells, which would be correlated with programmed cell death, was not observed. Mice lacking TGF-beta 3 did not have cleft mandible, but had clefting of the secondary palate as a feature of null mutation phenotype. We interpret our comparisons between wild type and homozygous TGF-beta 3 (-/-) mice to suggest that different developmental processes control palatal vs. mandibular fusion. We hypothesize that medical epithelial cells at the fusion site of mandibular prominences migrate to the surface epithelium during the fusion process and neither transdifferentiate into mesenchyme nor express apoptosis.

BCR/ABL has a causal role in Philadelphia (Ph)-chromosome positive leukemia. The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosine-phosphorylated in Ph-positive leukemia cells. In this study, experiments were pursued to identify and analyse the CRKL phosphorylation site(s). In an in vitro kinase assay, CRKL phosphorylation by the abl kinase was limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F did not transform NIH3T3 cells, while the Y207F mutation eliminated tyrosine-phosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL did not enhance or decrease association with various target signalling proteins, including SOS or C3G, which interact specifically with the CRKL N-SH3 domain. These findings suggest that complex formation with cellular targets is not modulated by CRKL tyrosine-phosphorylation.

Loss of heterozygosity on chromosome 17p13.3 is frequently observed in solid tumors, and the presence of a tumor suppressor gene has been predicted in this region of chromosome 17. We have analyzed a primitive neuroectodermal tumor sample exhibiting loss of heterozygosity at the D17S34 locus, a commonly used telomeric marker on the short arm of chromosome 17. The remaining allele showed a rearrangement. Cosmids spanning the D17S34 locus and probes from that region were used to demonstrate a 9-kb deletion within the D17S34 locus and were found to contain evolutionary, conserved sequences. Genetic alterations in this region may also affect expression of immediately adjacent genes, such as ABR, and could be a common mechanism in the causation of primitive neuroectodermal tumors.

Aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation in humans, is caused by mutations in the gene encoding the lysosomal enzyme glycosylasparaginase (Aga). The resulting enzyme deficiency allows aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines to accumulate in tissues and body fluids, from early fetal life onward. The clinical course is characterized by normal early development, slowly progressing to severe mental and motor retardation in early adulthood. The exact pathogenesis of AGU in humans is unknown and neither therapy nor an animal model for this debilitating and ultimately fatal disease exists. Through targeted disruption of the mouse Aga gene in embryonic stem cells, we generated mice that completely lack Aga activity. At the age of 5-10 months a massive accumulation of GlcNAc-Asn was detected along with lysosomal vacuolization, axonal swelling in the gracile nucleus and impaired neuromotor coordination. A significant number of older male mice had massively swollen bladders, which was not caused by obstruction, but most likely related to the impaired function of the nervous system. These findings are consistent with the pathogenesis of AGU and provide further data explaining the impaired neurological function in AGU patients.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190 constructs in transgenic mice. mRNA transcripts were detectable early in embryonic development and also in hematopoietic tissue of adult animals. Expression of BCR/ABL in peripheral blood preceded development of overt disease. P210 founder and progeny transgenic animals, when becoming ill, developed leukemia of B, T-lymphoid, or myeloid origin after a relatively long latency period. In contrast, P190-transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. The observed dissimilarities are most likely due to intrinsically different properties of the P190 and P210 oncoproteins and may also involve sequences that control transgene expression. The delayed progression of BCR/ABL P210-associated disease in the transgenic mice is consistent with the apparent indolence of human chronic myeloid leukemia during the chronic phase. We conclude that, in transgenic models, comparable expression of BCR/ABL P210 and BCR/ABL P190 results in clinically distinct conditions.

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.

A broad spectrum of biological activities has been proposed for transforming growth factor-beta 3 (TGF-beta 3). To study TGF-beta 3 function in development, TGF-beta 3 null mutant mice were generated by gene-targeting. Within 20 hours of birth, homozygous TGF-beta 3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF-beta 3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF-beta 3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial-mesenchymal interaction.

Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling.

The SH2/SH3 adaptor protein Crkl is abnormally phosphorylated on tyrosine by the Bcr/Abl protein in leukemic cells from patients with Philadelphia-chromosome (Ph)positive leukemia. However, the state of tyrosine-phosphorylation of crkl in normal tissues is unknown. In the current study, we identified mouse crkl by cDNA cloning and examined expression levels and tyrosine-phosphorylation of the mouse crkl protein during embryogenesis and in adult tissues. Tyrosine-phosphorylation of crkl was prominent during early development, but decreased at later embryonic stages and in newborn mice. Expression of both crkl and the related crk was ubiquitous in the adult. However, crkl differed considerably from crk in relative tissue distribution, and was more abundant in hematopoietic tissues. With exception of the lung, crkl was mostly present in a non-tyrosine phosphorylated form. Consistent with our previous findings in human patients, murine crkl was phosphorylated on tyrosine in leukemic tissues of BCR/ABL transgenic animals, but was non-tyrosine phosphorylated in normal mouse bone marrow. We conclude that this crkl tyrosine-phosphorylation by Bcr/Abl in hematopoietic cells is clearly aberrant and is consistently linked to the development of leukemia. Identification of proteins interacting with tyrosine-phosphorylated crkl in the leukemic cells of BCR/ABL transgenic mice should reveal members of signal transduction pathways activated in Ph-positive leukemia.

A high percentage of patients with DiGeorge syndrome and velo-cardio-facial syndrome have interstitial deletions on chromosome 22q11. The shortest region of overlap is currently estimated to be around 55 kb. Two segments of DNA from chromosome 22q11, located 160 kb apart, were cloned because they contained NotI restriction enzyme sites. In the current study we demonstrate that these segments are absent from chromosomes 22 carrying microdeletions of two different DiGeorge patients. Fluorescence in situ and Southern blot hybridization was further used to show that this locus is within the DiGeorge critical region. Phylogenetically conserved sequences adjacent to one human cell lines. cDNAs isolated with a probe from this segment showed it to contain the gene for teh human mitochondrial citrate transporter protein. Deletion of this gene in DiGeorge syndrome and velocardio-facial syndrome may contribute to the mental deficiency seen in the patients.

The deregulated tyrosine kinase activity of the Bcr/Abl protein has been causally linked to the development of Philadelphia (Ph) chromosome-positive leukemia in mice and man. Abnormally tyrosine-phosphorylated substrates of the Bcr/Abl kinase in Ph-positive cells are likely to contribute to leukemogenesis by interfering with normal signal transduction pathways. We have previously shown that the adaptor molecule Crkl is a major in vivo substrate for the Bcr/Abl tyrosine kinase, and it is thought to connect Bcr/Abl with downstream effectors. In the current study, a tyrosine-phosphorylated protein with a molecular mass of approximately 120 kDa was identified which binds only to the Crkl Src homology 2 (SH2) domain in cells, including Ph-positive patient material, containing an active Bcr/Abl protein. We demonstrate here that this protein is Cbl, originally discovered as an oncogene which induces B-cell and myeloid leukemias in mice. The Crkl SH2 domain binds specifically to Cbl. The Src homology 3 (SH3) domains of Crkl do not bind to Cbl, but do bind Bcr/Abl. These findings suggest the existence of a trimolecular complex involving Bcr/Abl, Crkl, and Cbl and are consistent with a model in which Crkl mediates the oncogenic signal of Bcr/Abl to Cbl.

Cytogenetic studies of Ph-positive leukemic patients and their parents have indicated that chromosome 22 involved in the formation of the t(9;22) is of maternal origin, whereas chromosome 9 is preferentially of paternal origin. These data have suggested that the two genes BCR and ABL, which become fused through the translocation, might be imprinted, ie expressed in a parental-specific manner. Recent molecular genetic studies however, have shown that BCR and ABL are expressed on both alleles and that the maternal and paternal ABL genes contribute equally often to the BCR-ABL fusion messenger. The findings make imprinting of these genes unlikely as an explanatory model and necessitate a combined cytogenetic and molecular genetic study.

Deletion of part or all of chromosome 17p is among the most frequent chromosome abnormalities in human cancer. We show that the CRK and ABR genes are close to a marker on chromosome 17p13.3, D17S34, which is frequently deleted in different tumours, and demonstrate that CRK is centromeric to ABR. CRK and ABR may be involved in cancer themselves, or otherwise may function as points of reference for further experiments to clone genes from chromosome 17p which may play a role in cancer.

Aspartylglycosaminuria (AGU) is the most common disorder of glycoprotein degradation. AGU patients are deficient in glycosylasparaginase (GA), which results in accumulation of aspartylglucosamine in body fluids and tissues. Human glycosylasparaginase was stably overexpressed in NIH-3T3 mouse fibroblasts, in which the unusual posttranslational processing and maturation of the enzyme occurred in a high degree. The recombinant enzyme was isolated as two isoforms, which were both phosphorylated, and actively transported into AGU fibroblasts and lymphoblasts through mannose-6-phosphate receptor-mediated endocytosis. The rate of uptake into fibroblasts was half-maximal when the concentration of GA in the medium was 5 x 10(-8) M. Immunofluorescence microscopy suggested compartmentalization of the recombinant enzyme in the lysosomes. Supplementation of culture medium with either isoform cleared AGU lymphoblasts of stored aspartylglucosamine when glycosylasparaginase activity in the cells reached 3-4% of that in normal lymphoblasts. A relatively small amount of recombinant GA in the culture medium was sufficient to reverse pathology in the target cells, indicating high corrective quality of the enzyme preparations. The combined evidence indicates that enzyme replacement therapy with the present recombinant glycosylasparaginase might reverse pathology at least in somatic cells of AGU patients.

The BCR gene is implicated in the development of Ph-positive leukemia through its fusion with the nonreceptor tyrosine kinase gene ABL. The normal 160 kDa Bcr protein has several functional domains, and recently one specific role for Bcr was established in the regulation of respiratory burst activity in white blood cells. Bcr expression levels are relatively constant throughout mouse development until adulthood in brain and in hematopoietic tissues, a pattern that is distinctly different from that of the functionally related n-chimerin gene. In the present study, RNA in situ hybridization was used to explore the normal cellular function of Bcr in rodent brain and hematopoietic organs. The data pinpoint the high bcr expression in the brain to the hippocampal pyramidal cell layer and the dentate gyrus, and to the piriform cortex and the olfactory nuclei, reflecting a potentially interesting function for Bcr in these highly specialized brain regions.

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.

Insulin and insulin-like growth factor-I (IGF-I) share a spectrum of metabolic and growth-promoting effects, mediated through homologous receptors that belong to the tyrosine kinase family. The dissociation rate of insulin from its receptor is affected by negative cooperativity, i.e. accelerates with increased receptor occupancy. The dose-response curve for the acceleration of tracer dissociation by unlabeled insulin has a distinct bell-shaped curve, with a progressive slowing down at insulin concentrations greater than 100 nM. The kinetics of the IGF-I interaction with its receptor has not been studied in such detail. In the present work, we report that while the IGF-I receptor exhibits negative cooperativity like the insulin receptor, the concentration dependence of the dissociation kinetics is distinct from that of native human insulin by not being bell-shaped, but monophasic like that of insulin analogues mutated at the hexamer-forming surface; it is changed to an insulin-type curve by substitution of IGF-I receptor's sequence including residues 382-565 with the homologous insulin receptor domain. The data suggest that like insulin, IGF-I has a bivalent binding mode and crosslinks two distinct areas of the two alpha subunits that are close, but distinct from the equivalent insulin receptor binding sites.

Interferon-alfa (IFN-alpha) is one of the most effective drugs in the treatment of chronic myeloid leukemia (CML). Recently, IFN-alpha has also been tried in the treatment of Ph-positive acute lymphoid leukemia (ALL), a disease in part sharing the same molecular genetic lesion as CML, namely a BCR/ABL fusion gene. In the present study we analyzed the effect of IFN-alpha (rHuIFN-alphaA/D) on a mouse model for Ph-positive ALL - mice transgenic for the P190 BCR/ABL fusion gene. IFN-alpha treatment was started in the early leukemic phase and continued throughout the course of the disease in eight transgenic animals. No prolonged survival or altered disease pattern with regard to the development of leukemia and/or lymphoma was observed. We conclude that IFN-alpha, at least in a transgenic setting, does not interfere with the leukemogenic process induced by the P190 BCR/ABL fusion gene.

Chronic myeloid leukemias and 5% to 20% of acute lymphoid leukemias are characterized by the Philadelphia chromosome, a reciprocal chromosomal translocation, t(9;22)(q34;q11), generating BCR-ABL and ABL-BCR fusion genes. Cytogenetic studies have recently shown a preferential involvement of the paternally derived chromosome 9 and the maternally derived chromosome 22 in this translocation, indicating that imprinting might be involved in the formation or selection of the translocation. In this study, we have identified a BamHI polymorphism in the coding region of BCR exon 1, allowing us to investigate whether both BCR alleles are transcribed. By using a reverse transcriptase-polymerase chain reaction assay, we show that both BCR alleles are expressed in the peripheral blood cells of normal individuals.

Chronic myelogenous leukemia is characterized by a specific chromosomal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chimeric BCR/ABL gene produces a P210 fusion protein with deregulated tyrosine kinase activity. We have recently isolated a complementary DNA, CRKL, which could code for an adaptor protein consisting of one SH2 and two SH3 domains and lacking any catalytic domain. In the current study, we show that CRKL is highly phosphorylated in the chronic myelogenous leukemia cell line K562 and that it is a substrate for the p210 BCR/ABL and p145 ABL kinases. BCR/ABL and ABL are coimmunoprecipitated with CRKL in vivo, demonstrating that relatively stable complexes are formed. In addition, the nucleotide exchange factor mSOS1 was found to be coimmunoprecipitated with CRKL. These findings establish a putative signal transduction pathway way through which BCR/ABL mediates its oncogenic activity.

In clinical and pharmacological laboratories, the assay for gamma-glutamyltransferase (GGT) activity is an important diagnostic test, but one with high biological variability. Although the human genome contains multiple GGT genomic sequences, the diagnostic tests generally assume that only a single GGT gene is active. In the current study, segments encompassing parts of seven different potential human GGT genes have been molecularly cloned. Based on sequence determination of exons within these distinct genomic clones, oligonucleotide primers were designed which would prime and PCR-amplify putative mRNA of all seven potential GGT genes, if expressed. Gene-specific oligonucleotide probes were then utilized to assay the transcriptional status of the seven possible GGT genes in a wide variety of human RNAs. Our results show that a single GGT gene exhibits ubiquitous expression in all RNAs tested, including those from fetal and adult liver. A surprisingly large number of four additional GGT genes is expressed in man. Interestingly, these novel GGT genes are expressed in a tissue-restricted manner, which suggests that their corresponding gene products exhibit distinct functions in these specific tissues.

Chromosome 22q11 contains a large number of interesting loci, including genes associated with cancer and developmental defects. The region is also the site of the lambda immunoglobulin variable and constant regions and the BCR, gamma-glutamyl transpeptidase, and GGT-like activity multigene families. Because of the complexities associated with mapping highly related gene families, we have examined the utility of mapping large areas of DNA using a defined approach. A total of 21 complete NotI sites from band q11 were cloned and ordered into six noncontiguous clusters of sites using a combination of somatic cell hybrid panels, NotI jumping and linking libraries, and fluorescence in situ hybridization. The largest cluster spanned an estimated 2 Mb of NotI fragments, the smallest 115 kb. Approximately 3.5 Mb of band q11 could be examined for rearrangements in NotI restriction enzyme fragments. A number of conserved sequences, two genes, and a minimum of two families of related sequences were identified adjacent to NotI sites.

A genomic DNA fragment, isolated from a human phage library using a chicken crk cDNA probe, was shown to derive from the human CRK locus. We have used fluorescent in situ hybridization (FISH) to map CRK distal in chromosome band 17p13, a region which demonstrates frequent deletion or loss of heterozygosity in a wide range of human cancers.

We have identified and partially characterized a gene located on chromosome 22, band q11, centromeric of the chronic myelogenous leukemia breakpoint region. A number of overlapping cDNAs were isolated from this locus and the largest of 1.8 kb was sequenced. Its deduced amino acid sequence shows homology to the SH2 domains of protein tyrosine kinases such as FER, and is strikingly similar to the cellular part of the v-crk oncogene product. We identified one SH2 and two SH3 domains within the 303 amino acid open reading frame of this crk-like gene, CRKL. The CRKL gene product is predicted to have a molecular mass of 36 kDa. In addition, we demonstrate that this gene does not represent the human homolog of v-crk but rather a novel gene potentially capable of mediating the transduction of intracellular signals.

It is well established that the chimeric BCR-ABL gene formed by joining parts of the BCR and ABL genes plays a key role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias. We report that simultaneous expression of P210 BCR-ABL and P160 BCR in simian COS-1 cells yielded stable complexes of these two proteins, and induced phosphorylation of P160 BCR on tyrosine residues in vivo. Tyrosine phosphorylation of a deletion mutant encoding 553 amino acids of BCR N-terminal sequences was also detected when it was coexpressed with P210 BCR-ABL. We propose that tyrosine phosphorylation of P160 BCR by P210 BCR-ABL and their stable physical interaction may perturb normal BCR functions and that these alterations are directly involved in the pathologic processes found in Ph chromosome-associated leukemias.

We have previously cloned a segment of a gene, ABR, homologous to the BCR gene, which encodes a protein consisting of three distinct functional domains. In the present study, genomic ABR sequences were used to isolate human ABR cDNAs. Surprisingly, the two types of ABR cDNAs identified differed only in their most 5' coding sequences. These are predicted to encode proteins of 93.5 and 92.3 kDa molecular mass. ABR showed a differential expression pattern in various mouse tissues, analogous to that of BCR, and the highest level was found in brain. Similar to BCR, ABR contains a region with homology to DBL, vav, and CDC24, which are likely to or have been shown to encode GTP exchange factors. A domain of ABR with similarity to GAPrho was expressed as a fusion protein in Escherichia coli and was shown to have GAP activity toward rac. Although both ABR and BCR have GAP activity, ABR lacks homology to the serine/threonine kinase domain of BCR. Therefore, ABR is likely to have cellular functions overlapping with but also distinct from those of BCR.

Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 microM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.

A gene family encompassing a minimum of four genes or pseudogenes for gamma-glutamyl transferase (GGT; EC 2.3.2.2) is present on chromosome 22q11. We have previously isolated a cDNA related to GGT but clearly not belonging to its gene family. The chromosomal location of this related gene, GGTLA1, has been determined by both isotopic and fluorescence in situ hybridization to metaphase cells and by Southern blot analysis of somatic cell hybrid DNAs. We show that GGTLA1 is part of a distinct gene family, which has at least four members (GGTLA1, GGTLA2, GGTLA3, GGTLA4). At least two loci are located on chromosome 22 within band q11 and proximal to the chronic myelogenous leukemia (CML) breakpoint in BCR (breakpoint cluster region gene). At least one other member is located more distally between the breakpoints found in Ewings sarcoma and CML. Some of the GGT and GGTLA family members are located on NotI restriction enzyme fragments of a similar size. Combined results indicate that a segment of human chromosome 22q11 has undergone large-scale amplification events relatively recently in evolution.

The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic mouse line. Over 95% of mice of this line die of leukemia or leukemia/lymphoma within 35-200 days of age. Karyotypically visible genetic alterations were absent from the early stages of BCR/ABL generated leukemia. A high frequency of aneuploidy was found in advanced leukemia indicating a primary and pivotal role for BCR/ABL in leukemogenesis. Moreover, the data suggest that BCR/ABL has a destabilizing effect on the regulation of the cell cycle. BCR/ABL expression was also found in tissues other than hematopoietic cells. However, this did not result in the development of solid tumors, strongly suggesting that the oncogenicity of BCR/ABL is limited to the hematopoietic lineage.

A chimeric BCR/ABL oncogene encoding the p190 protein has been introduced into the mouse germline using microinjection of one-cell fertilized eggs. Founder and progeny transgenic animals, when becoming ill, were found to develop lymphoblastic leukemia/lymphoma which was transplantable to compatible recipients. Lymphoblasts were arrested at the pre-B stage of development. Expression of BCR/ABL was not detected in peripheral blood during the early stages of leukemia but became evident as the disease progressed. However, the transgene was expressed early in development in bone marrow and was also transcribed in nonhematopoietic tissues although this did not result in tumorigenesis. These results strongly suggest that the oncogenicity of BCR/ABL is limited to hematopoietic cells, including pre-B cells or their progenitors.

The clonal and immunophenotypic characteristics of blood leukemic cells from BCR/ABL p190 transgenic mice were investigated. All cell populations evaluated in vivo and in vitro had B-lymphocyte progenitor immunophenotypes. Immunoglobulin (JH) rearrangement patterns provided evidence for clonal diversification at different sites in vivo. Multiple clones were established in vitro from two of these mice (nos. 730 and 753). These cells expressed BCR/ABL p190 protein tyrosine kinase (PTK) and were highly malignant on transfer to secondary recipients. Cells independently cloned in vitro shared identical immunophenotypes and clonal IgH rearrangements, but these were distinct from those of the dominant clones in the mouse from which they were derived. Nevertheless, in vitro clones from mouse no. 753 had an abnormal karyotype (chromosome 14 trisomy) in common with the dominant clone in blood, providing evidence for a hierarchy or clonal selection in vivo and in vitro. Two sets of in vitro clones proliferated independently of exogenous growth factors and stroma and released autocrine interleukin 7 growth factor activity. These data provide evidence for rapid divergent clonal evolution and selection of B-cell progenitors initiated by BCR/ABL p190, followed by other, secondary genetic events mirroring similar changes in the equivalent, highly malignant human leukemia Philadelphia (Ph)-positive/B-precursor acute lymphoblastic leukemia (ALL).