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Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ “cancer stem cells,” such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000–50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-β-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF.elf+/−mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-β signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC inelf+/−mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-β signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-β pathway.

Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P

Mitogen-activated protein kinases (MAPKs) regulate critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Recent studies have shown that a novel class of scaffold proteins mediates the structural and functional organization of the three-tier MAPK module. By linking the MAP3K, MAP2K and MAPK into a multienzyme complex, these MAPK-specific scaffold proteins provide an insulated physical conduit through which signals from the respective MAPK can be transmitted to the appropriate spatiotemporal cellular loci. Scaffold proteins play a determinant role in modulating the signaling strength of their cognate MAPK module by regulating the signal amplitude and duration. The scaffold proteins themselves are finely regulated resulting in dynamic intra- and inter-molecular interactions that can modulate the signaling outputs of MAPK modules. This review focuses on defining the diverse mechanisms by which these scaffold proteins interact with their respective MAPK modules and the role of such interactions in the spatiotemporal organization as well as context-specific signaling of the different MAPK modules.

During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kappaB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kappaB. We conclude that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers.

The v-src oncogene encodes a nonreceptor tyrosine kinase. When this gene was expressed in the myeloblastic cell line 32Dcl3, it was found to abrogate interleukin-3 (IL-3) dependence of this cell line and to block its ability to terminally differentiate into granulocytes in response to granulocyte colony-stimulating factor (GCSF). In contrast, a highly related tyrosine kinase gene, v-fgr, fails to render this cell line IL-3 independent for growth or to block its ability to undergo terminal differentiation in the presence of GCSF. The active structural domains of v-src that are responsible for the abrogation of IL-3 dependence of myeloid cells and the mechanisms by which v-src transforms these cells are at present unclear. To identify the domains in v-src which are responsible for this activity, we constructed several chimeric recombinants between the v-src and the related Src family member v-fgr by replacing portions of v-src with corresponding domains of v-fgr. These chimeric DNAs were transfected into 32Dcl3 cells and examined for their abilities to render this cell line IL-3 independent. Our results show that only chimeras containing both the SH3 and the SH2 domains of v-src were capable of rendering the 32Dcl3 cell line IL-3 independent. To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dcl3 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dcl3 cells. Our results show that none of the JAK kinases are constitutively phosphorylated by v-src or v-fgr. We then examined the phosphorylation status of the STAT (signal transducers and activators of transcription) family of transcription factors. Our results show that STAT1, STAT3, and STAT5 exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, while such constitutive phosphorylation is not seen in v-fgr-transformed cell lines. Our results also show that STAT3 coimmunoprecipitates with v-Src, suggesting that the activation of STAT3 occurs due to direct association with v-Src. However, STAT1 and STAT5, which also exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly with v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.

To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.

The pupose of this study was to evaluate the activity of ON 01910.Na, a mitotic inhibitor, in in vitro and in vivo models of pancreatic cancer and to discover biomarkers predictive of efficacy. Successive in vitro and in vivo models were used; these included cell line-derived and patient-derived tumors from our PancXenoBank, a live collection of freshly generated pancreatic cancer xenografts. ON 01910.Na showed equivalent activity to gemcitabine against pancreatic cancer cell lines in vitro. The activity of the agent correlated with suppression of phospho-CDC25C and cyclin B1. These markers were optimized for a fine-needle aspirate ex vivo rapid assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Next, nine patient-derived tumors from the PancXenoBank were profiled using the assay developed in cell lines and treated with ON01910.Na for 28 days. Two cases were cataloged as potential responders and seven as resistants. There was a correlation between the ex vivo assay and sensitivity to the tested agent, as the two cases prospectively identified as sensitive met prespecified criteria for response. Of the seven tumors of predictive resistant, only one was found to be sensitive to ON 01910.Na. In addition, there was a good correlation between cyclin B1 downregulation ex vivo and changes in cyclin B1 protein post-treatment. The novel mitotic inhibitor, ON 01910.Na, showed activity in preclinical model of pancreatic cancer. A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure.

Activation of the c-abl protooncogene occurs in Abelson murine leukemia virus, in Hardy-Zuckerman 2 feline sarcoma virus, and during the chromosomal translocations that generate BCR-ABL gene fusion products. To study the molecular mechanism involved in the c-abl activation, we have created a series of modifications in murine c-abl and assayed these constructs for oncogenic activity using the NIH 3T3 cell transformation assay. Our results show that amino-terminal deletions are sufficient for oncogenic activation of c-abl and high levels of oncogenic activities were generated by a deletion of 114 codons from the 5' end that deleted the SH3 region. A deletion of 53 codons from the 5' end (inclusive of deletions seen in Hardy-Zuckerman 2 feline sarcoma virus and BCR-ABL gene products) that retains the SH3 region of c-abl resulted in the generation of low levels of transforming activity. This transforming potential was substantially increased with the introduction of a G----A point mutation in codon 832 that is present in v-abl. The point mutation was found to affect the secondary structure and the tyrosine kinase activity of the mutant gene products.

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Proper culmination of these diverse pathways forms the basis for an orderly generation of different cell types. Recent studies conducted over the past 10-15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors termed STATs (signal transducers and activators of transcription). Aberration in these pathways, such as that caused by the recently identified JAK2V617F mutation, is an underlying cause for diseases such as leukemias and other myeloproliferative disorders. This recent discovery, when coupled with the fact that STATs are activated by oncoproteins such as BCR-ABL, underscores the importance of the JAK-STAT pathway in both normal cellular development and disease states.

Inactivation of the transforming growth factor-beta (TGF-beta) pathway occurs often in malignancies of the gastrointestinal (GI) system. However, only a fraction of sporadic GI tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Here, we show a wide range of GI tumors, including those of the stomach, liver and colon in elf+/- and elf+/- / Smad4+/- mutant mice. We found that embryonic liver fodrin (ELF), a beta-Spectrin originally identified in endodermal stem/progenitor cells committed to foregut lineage, possesses potent antioncogenic activity and is frequently inactivated in GI cancers. Specifically, E-cadherin accumulation at cell-cell contacts and E-cadherin-beta-catenin-dependent epithelial cell-cell adhesion is disrupted in elf+/- / Smad4+/- mutant gastric epithelial cells, and could be rescued by ectopic expression of full-length elf, but not Smad3 or Smad4. Subcellular fractionation revealed that E-cadherin is expressed mainly at the cell membrane after TGF-beta stimulation. In contrast, elf+/- / Smad4+/- mutant tissues showed abnormal distribution of E-cadherin that could be rescued by overexpression of ELF but not Smad3 or Smad4. Our results identify a group of common lethal malignancies in which inactivation of TGF-beta signaling, which is essential for tumor suppression, is disrupted by inactivation of the ELF adaptor protein.

We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.

Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals.

Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphorylation during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no effect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not affect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphorylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide antiapoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of which results in proliferation and survival of myeloid precursor cells.

Apoptosis, or programmed cell death, is a process where developmental or environmental stimuli activate a genetic program to implement a series of events that culminate in cell death. To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, we utilized the 32Dcl3 cell line, which is derived from normal mouse bone marrow, is non-tumorigenic and diploid. These cells are strictly dependent on IL-3 for growth and apoptose when deprived of IL-3. However, when these cells are transferred to medium containing G-CSF, the cell number increases 4 – 5-fold and after 12 days the entire population is differentiated into granulocytes followed by apoptotic death. In our search for genes that are induced during apoptosis and/or terminal differentiation of 32Dcl3 cells, we identified a novel gene termed AATYK (Apoptosis Associated Tyrosine Kinase), whose expression is dramatically upregulated during IL-3 deprivation as well as G-CSF-induced terminal differentiation. In this report, we describe the sequence of the cDNA clone, derived from the mRNA transcript of this gene. These studies show that this gene encodes a protein with a tyrosine kinase domain at the N-terminal end and a proline-rich domain at the C-terminal end. We also report that the expression of this gene is blocked in v-abl or bcr – abl transformed myeloid cells which are unable to apoptose when grown in the absence of IL-3. However, AATYK expression is induced in 32D cells transformed by the v-abl gene when these cells are incubated in the presence of DMSO, which induces growth arrest and apoptotic death of the cells. On the other hand, DMSO fails to induce apoptosis or AATYK expression in 32D cells transformed by the bcr – abl oncogene, suggesting that AATYK expression may be a necessary pre-requisite for the induction of growth arrest and/or apoptosis of myeloid precursor cells.

The Myb gene family currently consists of three members, named A-, B- and c-myb1,2. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells2,3. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb−/− males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.

In this study, we have isolated the genomic DNA clone encoding the murine JAK3 gene and determined its sequence. Partial genomic clones of the JAK1 and JAK2 genes encoding the tyrosine kinase domain were also isolated and compared with JAK3. The genomic structure of JAK3 consists of 23 exons. The exon/intron boundaries and the distribution of coding sequences within the exons of each of the three JAK genes were found to follow a similar pattern suggesting that various members of the JAK family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. Using in situ hybridization and FISH analysis, we have mapped the JAK3 gene to human chromosome 19p13.1-p13.2.

We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.

The myb gene has been shown to be an important regulator of hematopoietic cell proliferation, differentiation and apoptosis. Activation of the myb gene into an oncogenic form has involved structural alterations to the coding sequences. Thus, the v-myb gene encoded by the Avian Myeloblastosis Virus, is truncated at both the 5' and 3' ends. Additionally, tumor cells containing rearrangements in the myb locus, such as the ABPL tumors or NFS60 tumor cell line have recently been shown to display a heterogeneity of structure. In this study, we examined the growth and differentiation properties of clonal cell lines derived from 32Dcl3 which harbor myb transgenes; derived from v-myb, and the ABPL-1, ABPL-2, ABPL-4 and NFS-60 cell lines. Retroviral vectors containing the appropriate myb cDNAs were produced, transfected into packaging cell lines, and the viruses were used to generate the 32D derivative cell clones. Abrogation of IL-3 dependence was never observed in any cell line. Expression of c-myb, ABPL-1-myb and ABPL-2-myb isoforms in 32D cells resulted in a block to their ability to terminally differentiate into granulocytes at the pro-myelocytic stage. However, expression of ABPL-4-myb or NFS60-myb in these cells failed to result in a similar effect. These cells differentiated into granulocytes in the presence of G-CSF, albeit more slowly than control 32Dcl3 cells. We also examined the ability of various Myb-isoforms to transactivate transcription of reporter genes containing Myb-binding elements in their promoter/enhancer sequences, to determine whether the phenotypic effects produced by these various isoforms correlate with their ability to transactivate transcription. Our results show that while v-myb and c-myb transactivated transcription equally well, the NFS60-myb exhibited the highest levels of transcriptional transactivation. The ABPL-1, ABPL-2 and ABPL-4-myb isoforms showed very low levels of transcriptional transactivation potential with the same reporter genes. These results suggest that the ability of various Myb-isoforms to transactivate transcription does not by itself correlate with their ability to induce a block to G-CSF-induced terminal differentiation of myeloid precursor cells.

Signal transduction pathways which are initiated by members of the TNF superfamily utilize receptors which are devoid of intrinsic catalytic activity. Isolation and characterization of death domain (TNF-RI, Fas, TRADD, FADD/MORT-1, RIP) and TRAF domain-containing proteins (TRAF-1, TRAF-2, TRAF-3) have partially bridged a large molecular gap within one of several signaling pathways which originate at the plasma membrane and terminate in the nucleus. The ability of these two protein families to selectively dimerize and bind to related receptors allows them to govern diverse cellular responses which culminate in cellular proliferation, differentiation, effector functions, and apoptosis.

Transcriptional regulation of lineage specific genes has the ability to dictate both the proliferative and differentiative potentials of a pluipotent precursor cell. The E2A and Pax5/BSAP genes encode transcription factors which bind to B cell specific promoters and enhancers and guide the development of immature, but committed cells into mature B lymphocytes which express and secrete immunoglobulins. In vitro analysis has consistently suggested that these proteins regulate distinct classes of genes during B cell differentiation; however, recent targeted gene disruption and transgene expression in mice has indicated that these genes may actually be components of a single regulatory mechanism which is essential for both B lymphocyte differentiation and proliferation.

All eukaryotic cells possess similar mechanisms to regulate the progression of the cell cycle. However, higher eukaryotes have evolved to respond to a large array of positive and negative signals with an intracellular or extracellular origin. These signals are eventually integrated by a conserved protein engine consisting of holoenzymes with kinase activity, which trigger crucial transitions during the cell cycle. In this review, the mechanisms by which the mammalian cell cycle engine integrates intracellular and extracellular signals of different nature are discussed.

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

The myb gene family consists of three members, A-myb, B-myb and c-myb. The proteins encoded by these genes bind DNA in a sequence-specific manner and regulate transcription of target genes. In this communication, we report the nucleotide sequence of murine A-myb cDNA. This gene encodes a protein of 751 amino acids with an estimated molecular weight of 83 kDa. cDNA sequence analysis of multiple independent cDNA clones reveals the presence of alternatively spliced mRNAs that encode smaller proteins. Northern blot analysis of adult mouse tissue RNAs reveals A-myb expression predominantly in the testis, with very low levels of expression in the ovaries, spleen and brain. In situ hybridization analysis of adult mouse testis shows that this gene is expressed at high levels in type A spermatogonia (stem cells), and preleptotene and pachytene spermatocytes, with concomitant down-regulation of expression upon terminal differentiation of these cells into mature spermatozoa. This pattern of expression suggests that A-myb is involved in the control of proliferation and differentiation of spermatogonia. This potential function of A-myb in spermatogenesis is reminiscent of the role of c-myb in hematopoiesis.

The Janus Kinases (JAK) JAK1, JAK2, and TYK2 are protein tyrosine kinases which play a pivotal role in the signal transduction process mediated by cytokines. These kinases appear to transduce signals via their substrates which modulate programs of gene expression specific to the respective signals. It is becoming increasingly evident that certain cytokines such as Granulocyte Colony Stimulating Factor (GCSF) can transmit signals for both cellular proliferation and differentiation. It is at present unclear whether both of these signals are transmitted by the same JAK kinase or whether an entire family of such kinases are involved in this process. To determine if additional members of JAK kinase family exist, we designed a polymerase chain reaction based strategy which resulted in the identification of a new member of the JAK kinase family. This new kinase, which we have named JAK3 is encoded by a 4.3 kb mRNA transcript. Nucleotide sequence analysis of a full length cDNA derived from this mRNA revealed that it encodes an open reading frame of 3897 bp. The protein encoded by this mRNA contains the double catalytic domain characteristic of the JAK family kinases. The most striking difference between JAK3 and the other JAK kinases is the presence of two stretches of additional amino acid sequence of 147 and 28 residues which span between amino acid positions 322 to 469 and 632 to 660 respectively. Expression studies indicate that JAK3 is expressed at very low levels in immature hematopoietic cells, but its expression is dramatically up-regulated during terminal differentiation of these cells. These results suggest that JAK3 plays an important role in the differentiation of hematopoietic cells.

Activation of the c-abl protooncogene occurs in Abelson murine leukemia virus, Hardy-Zuckerman-2 feline sarcoma virus, and during the chromosomal translocation that generates the BCRABL fusion gene. The three genes exhibit varying degrees of transforming activity; the two viral genes transform NIH-3T3 cells in vitro, whereas the BCRABL gene is incapable of transforming these cells. To determine whether genetic alterations can enhance the transforming potential of the BCRABL gene, we employed genetic selection techniques which led to the isolation of a mutant form of the BCRABL gene with high levels of fibroblastic transforming activity. Molecular analysis of this clone shows that it suffered a deletion of 3' ABL sequences and their replacement with a cellular sequence of unknown origin, termed X. This tripartite gene is capable of inducing 35 foci/10 ng of DNA. Deletion of 3' ABL sequences analogous to that seen in the activated BCRABL protein without the addition of X yields 5 foci/100 ng of DNA. These results suggest that carboxyl-terminal truncations unmask the fibroblastic transforming activity of the BCRABL gene product and the addition of X sequences dramatically enhances this transforming potential, indicating a dominant contribution by the X reading frame.

The c-myb gene products are thought to be regulators of cellular replication and of differentiation and heterogeneity may underlie their multiple functions. To investigate the possible existence of heterogeneity we have examined the chicken c-myb mRNAs by Northern blot analysis and polymerase chain reaction amplification of cDNAs (RT-PCR). Northern blot analysis with the c-myb cDNA clone pSG3, which contains the entire open reading frame (ORF) plus 500 base pairs of 3' untranslated sequences (GerondakisBishop, 1986), and genomic probes revealed c-myb RNA species of 4.3 kb in addition to the major 4.0 kb species. The 4.3 kb c-myb RNA contained the alternatively spliced exon 9A which is highly conserved and has also been detected in a minor 4.3 kb alternatively spliced c-myb mRNA in murine and human cells. Sequencing of the avian exon 9A revealed 360 bp exon homologous to that found in murine and human mRNAs, which contains three highly conserved sequence regions shared by all three species. RT-PCR demonstrated usage of exon 9A in five hematopoietic tissues and revealed an additional splicing variant which used the 3' portion of exon 9A. Northern blot analysis using splice site-specific oligonucleotide probes spanning the two splice junctions between exon 9 and 9A revealed four additional c-myb RNAs of 4.4 kb, 2.2 kb, 2.0 kb and 1.4 kb.

Diabetes mellitus ensues as a consequence of the body's inability to respond normally to high blood glucose levels. The onset of diabetes is due to several pathological changes, which are a reflection of either the inability of the pancreatic beta cells to secrete sufficient insulin to combat the hyperglycemia or a state of insulin resistance in target tissues. However, the significance of changes in beta cell mass and decreased beta cell proliferation or growth in progression of diabetes has been under-appreciated. Beta cells, like all other cells of our body are under the regulatory checks and balances enforced by changes in cell cycle progression. However, very little is known regarding the key components of the cell cycle machinery regulating cell cycle control of beta cells. Knowledge of key elements involved in cell cycle regulation of beta cells will go a long way in improving our understanding of the replication capacity and developmental biology of beta cells. This information is essential for us to design new approaches that can be used to correct beta cell deficiency in diabetes. This review focuses on the current knowledge of factors important for proliferation of beta cells and proposes a cell cycle model for regeneration of the beta cell population lost or reduced in diabetes.

Activation of the abl gene and its involvement in human leukemia is one of the most thoroughly characterized examples of the structural alterations of chromosomes associated with the conversion of a normal cell into a cancer cell. The abl oncogene as first identified on the Abelson murine leukemia virus (A-MuLV). Activation of the viral oncogene is associated, in part, with the truncation of the gene at its 5' end. As in studies with other retroviruses, results with A-MuLV presaged the mechanism of activation by abl in naturally occurring human malignancies. Thus, chronic myelogenous leukemia (CML) is consistently associated with a translocation of a piece of chromosome 9 onto chromosome 22 creating what is known as the Philadelphia chromosome (Ph1). The result of this translocation is the truncation of the 5' end of the cellular abl gene, which is located at the breakpoint of chromosome 9. The function of the abl gene product is poorly understood but is thought to participate in an, as yet, undefined pathway of growth control signals, which originate outside the cell, and traverse through the cell into its nucleus. The loss of the gene product's N-terminal amino acid sequences brought about by the truncation of the 5' portion of the gene is consistent with the hypothesis that the protein's growth-controlling activity is deregulated by the structural alterations which occur in the cancer cells. The abl gene and CML serve as a paradigm of the mechanism of activation of proto-oncogenes by chromosomal alterations. The case of CML and the Ph1 chromosome illustrates the findings we might expect as other chromosomal abnormalities are characterized at the molecular level.

The integrated proviral genome of Abelson murine leukemia virus (A-MuLV) was cloned in lambda gtWES . lambda B bacteriophage after EcoRI endonuclease digestion and enrichment of proviral sequences by sequential RPC-5 column chromatography and agarose gel electrophoresis. Recombinant DNA clones containing a 7.8-kilobase-pair EcoRI insert were shown to have the entire integrated A-MuLV genome with both 5' and 3' ends flanked by mink cellular DNA sequences. This DNA fragment was shown to induce focus transformation upon transfection of NIH/3T3 mouse cells. Moreover, focus-forming virus could be rescued from transformed nonproducer cells upon superinfection with a type C helper virus. A polyprotein of molecular weight 120,000 (p120) containing murine leukemia virus gag gene determinants was invariably deteced by immunoprecipitation analysis of individual transformants induced by the 7.8-kilobase-pair DNA. Molecularly cloned integrated A-MuLV in its infectious form should be of use in elucidating the mechanisms involved in transformation by this virus.

Among a series of myeloid leukemia cell lines, one (NFS-60) was found to have a rearrangement of the c-myb locus. The rearrangement involved the integration of a retrovirus into the region of the gene corresponding to the sixth exon of the avian c-myb locus. The insertion is associated with the production of a truncated RNA and the introduction of a terminator codon at the juncture of the long terminal repeat and the c-myb locus. The properties of the NSF-60 cells were compared with those of other myeloid cell lines, and the known sequence of differentiation induced by interleukin 3. Similar to other myeloid cell lines, the NFS-60 cells do not terminally differentiate in response to interleukin 3, granulocyte/macrophage, or granulocyte colony-stimulating factor suggesting that the cells are transformed with regard to their ability to differentiate. The NFS-60 cells are totally dependent on interleukin 3 for growth and maintenance of viability in vitro but also proliferate in response to granulocyte colony-stimulating factor. The properties of the cells support the concept that the c-myb protooncogene is involved in the control of normal differentiation of hematopoietic cells.

The nucleotide sequence of the proviral genome of Abelson murine leukemia virus (A-MuLV), an acute transforming virus of murine origin, has been determined. Like other transforming viruses, A-MuLV contains sequences derived from its helper virus, Moloney murine leukemia virus (M-MuLV), and a cell-derived protooncogene (abl) insertion sequence. By comparison of the A-MuLV sequence with that of M-MuLV, it was possible to precisely localize and define sequences contributed by the host cellular DNA. From the nucleotide sequence, we have predicted the amino acid sequence of p120gag-abl, the product of the A-MuLV gag-abl hybrid gene. The amino acid sequence of the putative abl gene, when compared with the sequences of other tyrosine-specific protein kinases (src, fes, fps, and yes), revealed significant homologies, indicating that all these functionally related transforming genes are derived from divergent members of the same protooncogene family. In addition to the gag-abl sequence, the proviral genome was found to contain an additional open reading frame that could code for an 18,000-dalton protein, whose role is at present undetermined.

The molecular mechanisms that modulate c-myb mRNA levels in hematopoietic cells appear to involve premature termination of transcription in the first intron of the gene. We have examined the DNA-protein interactions within the first intron of the c-myb gene and identified a 1.0-kilobase region that could be responsible for its transcriptional regulation. Using the mobility-shift assay, we show a direct correlation between the extent of sequence-specific protein binding to intron 1 DNA fragments, and c-myb mRNA levels in different cell types. During dimethyl sulfoxide-induced differentiation of mouse erythroleukemic cells, there was a dramatic decrease in these nuclear factors that correlated with the decrease in the levels of c-myb mRNA. Nucleotide sequence analysis and DNase I footprinting revealed the presence of putative regulatory elements that are implicated in the binding of these nuclear factors. We propose that binding of nuclear factors to the site of transcriptional pause could play an important role in the regulation of c-myb transcription.

The c-abl gene codes for a protein-tyrosine kinase and is expressed in most examined murine cell types as two distinct mRNA species of 5.5 kilobases (kb) and 6.5 kb. In mouse testis, an additional species of 4.0 kb is expressed in very high levels. To study the interrelationship between various c-abl transcripts and to compare their sequence with the v-abl transcript, we prepared c-abl-specific cDNA clones from mouse testis and determined the complete nucleotide sequence of the 4.0-kb cDNA that appears to be the reverse transcript of the testis-specific mRNA. In addition, we have determined the 3' sequence of an additional clone derived from the larger mRNA species that is expressed in somatic as well as germ-line cells. These cDNA sequences have been compared with the v-abl sequences to understand the mechanism of activation of this oncogene. The results demonstrate that (i) testis-specific c-abl mRNAs arise as a result of 3' truncation, and (ii) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.

RNA blots of poly(A)-containing RNA from normal livers and spleens and from a number of transplantable hematopoietic and lymphoid BALB/c tumors, including early and late generation plasmacytomas, were hybridized with probes for four onc genes. abl RNA was abundant only in those tumors producing Abelson virus, bas RNA was found in approximately equal amounts in normal tissues and plasmacytomas, and myb RNA was absent in normal liver and plasmacytomas. Normal liver and spleen RNA showed faint traces of myc hybridization, but myc RNA was increased in most plasmacytomas. In one plasmacytoma, TEPC 1165, a particularly abundant amount of myc RNA was found, principally as a 3.5-kilobase band. In the other plasmacytomas, bands of 2.4- or 1.8-kilobase myc RNA were found. Southern blots of DNA from tumors that contained 2.4-kilobase or larger myc RNA showed myc hybridization to an EcoRI fragment of about 21 kilobase pairs, similar to the myc band in normal DNA. EcoRI digests of DNA from two tumors that expressed myc RNA of 1.8 kilobases showed an additional smaller myc band, suggesting that the myc gene is rearranged in these plasmacytomas. The basis for increased myc gene transcription in plasmacytomas is not understood, but the evidence suggests that different mechanisms may be operating in different plasmacytomas. Apparently, neither myc gene amplification nor myc gene rearrangement is required for increased myc transcription.

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.

It has been recently shown that malignant activation of the c-has/bas proto-oncogene in T24 human bladder carcinoma cells was mediated by a single point mutation. A deoxyguanosine located at position 35 of the first exon of this proto-oncogene was substituted by thymidine. These findings predicted that the resulting oncogene would code for a structurally altered p21 protein containing valine instead of glycine as its 12th amino acid residue. We now report the spontaneous activation of the human c-has/bas proto-oncogene during transfection of NIH/3T3 cells. As in T24 cells, this in vitro activated oncogene also acquired malignant properties by a single point mutation. In this case we have detected a G leads to A transition, which occurred at the same position as the mutation responsible for the activation of the T24 oncogene. These results predict that the p21 protein coded for by the spontaneously activated c-has/bas gene will incorporate aspartic acid as its 12th amino acid residue. Computer analysis of the secondary structure of c-has/bas encoded p21 proteins indicates that substitution of the glycine residue located at position 12, not only by aspartic acid or valine but also by any other amino acid, would result in the same structural alteration. These findings indicate that a specific conformational change is sufficient to confer transforming properties to this p21 protein. Moreover, they predict that any mutation affecting the coding properties of the 12th codon of the c-has/bas proto-oncogene will lead to its malignant activation.

Lymphoid cells transformed by Rauscher murine leukemia virus (R-MuLV) belonged to the B cell lineages. One group of cells exhibited Fc receptors but completely lacked immunoglobulin mu heavy and kappa light chains. The majority of the cells resemble pre-B type. They displayed mu chains but kappa chains were completely absent. Very rarely certain cells synthesized both mu and kappa chains. Based on the presence of Fc receptors and IgM synthesis the cells transformed by R-MuLV belonged to three B cell developmental stages. These cells were tested for immunoglobulin gene rearrangements using JH and CK probes. DNA from cell lines without any detectable levels of IgM mu exhibited embryonic as well as rearranged JH genes, whereas cells expressing IgM possess, in addition, productive and non-productive light chain gene rearrangements. The most terminally differentiated cell possesses JH and CK rearrangement associated with the synthesis of mu and kappa chains. Presumably the cells with rearranged JH and CK genes without immunoglobulin synthesis represent a developmental transition. We conclude that cells transformed by R-MuLV belonged to five step-wise compartments of B cell development. Our findings implicate definite sequential events of immunoglobulin gene rearrangement and expression during B cell development.

There are only four positions in the amino acid sequences of immunoglobulin variable region domains of all species at which the same amino acid is always found1. The compiled sequences of rabbit heavy chain variable (VH) domains have 48 invariant positions1,2 and at 14 additional positions in the first and third framework segments the different amino acids found correlate with the rabbits' VHa allotype3–5. This VH allotypic system with three common ‘alleles’ VHa1, VHa2 and VHa3 inherited in simple mendelian fashion3,4 poses the question of how gene families with distinct sets of conserved codons have evolved and been maintained in the midst of information leading to hypervariability. Here we report a cDNA sequence that encodes a protein characteristic of the rabbit VHa2 allotype and we predict the allotype-associated codons that may be found in VHa1 and VHa3 genes. We have also found within the second hypervariable, complementarity-determining region (CDR2) encoded by our cDNA clone, a DNA sequence highly homologous to a human DH minigene6. This observation and a similar one of Wu and Kabat7, who identified a DH minigene sequence in the CDR2 of a cloned human VH gene8, lends support to their idea that D minigene information has contributed to CDR2 either during evolution or ontogeny7. Gene conversion9,10, minigene assortment11,12 or insertion13 mechanisms may be involved both in the generation of hypervariability and the conservation of allotype codons.

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.

Simian sarcoma virus (SSV) deletion mutants were constructed from a molecular clone containing the entire infectious provirus. Transfection analysis of these mutants localized the SSV transforming gene to a small region of the viral genome encompassing its cell-derived sequence (v-sis). Antiserum to a peptide synthesized on the basis of the predicted amino acid sequence of the SSV transforming gene detected a 28,000-dalton protein that was specifically expressed in SSV transformed cells and that corresponded in size to that predicted from the v-sis coding sequence. The v-sis gene product designated p28sis was not a phosphoprotein, nor did it possess detectable protein kinase activity. These findings distinguish p28sis from a number of other retroviral onc proteins.

The genome of the replication-defective avian myeloblastosis virus (AMV) contains an inserted cellular sequence (amv) that is part of the oncogene responsible for acute myeloblastic leukemia in chickens infected with AMV. Three antisera raised against distinct synthetic peptides predicted from the long open reading frame of amv specifically precipitated the same 48-kilodalton protein (p48amv) from leukemic myeloblasts but not from normal hematopoietic tissue, fibroblasts, or from fibroblasts infected with the AMV helper virus, MAV-1 (myeloblastosis-associated virus type 1). p48amv is not glycosylated or phosphorylated and does not appear to act as a protein kinase in vitro. The same three antisera that recognized p48amv also specifically precipitated a common 110-kilodalton protein from normal uninfected hematopoietic tissue. This normal cellular homologue of the AMV leukemogenic protein, p110proto-amv, was not present in normal fibroblasts, MAV-1 infected fibroblasts, or, interestingly, in some leukemic myeloblasts. We conclude that p48amv is the leukemogenic product of an altered, transduced, partial protooncogene. Short helper-virus sequences provide its carboxyl terminus and also may provide the amino terminus.

We have optimized the conditions for efficient NIH3T3 focus formation by calcium phosphate transfection of proviral Abelson-murine leukemia virus (A-MuLV) plasmid DNA. Linearized pA-MuLV, P120 or P160 strains, when transfected with calf thymus carrier DNA, will produce 40-50 foci/100 ng pA-MuLV without co-transfecting Moloney-murine leukemia virus (Mo-MuLV) plasmid DNA.

Activation of the mouse c-myb oncogene in Abelson virus-induced plasmacytoid lymphosarcomas was studied using cDNA cloning and nucleotide sequence analysis. The results presented here show that viral integration in the myb locus generates splicing errors at the 5' and 3' regions. Viral integration results in transcriptional initiation within the viral long terminal repeat and generation of a chimeric mRNA that lacks the first three coding exons. The alterations at the 3' end are caused by an aberrant splicing event in which additional splice-donor and -acceptor sequences within intronic sequences are used to splice an additional 363 nucleotides into the myb transcripts. The resulting insertion of 121 amino acids is in a region of the protein where other activated forms of the myb gene product have deletions. These results suggest that alterations in the 3' end of the myb gene play a crucial role in the activation of this gene.

The allelism of the structural genes for the complex rabbit b allotypes of immunoglobulin kappa-light chains has been questioned because of observations of unexpected phenotypic expression of "latent" allotypes. We find that the coding sequences of the b4 and b5 "alleles" are only 80% homologous for the last 60 nucleotides but there is a high degree of homology (96%) in the 3' untranslated region (3'DT). The high conservation of 3' DT region sequences enabled us to detect kappa-light chain mRNAs from rabbits of different genetic types (b4, b5, b9 and bbas) on northern blots and dot blots. We can distinguish mRNA encoding b9 and b5 allotypes on dot blots with b5 fragment-probes of known sequence and detect mRNA produced by unstimulated cultured splenic lymphocytes. Analyses of mRNA from cultured cells manipulated to enhance mRNA synthesis and production of unexpected or "latent" b allotypes can now be conducted.

Rearrangement in the c-myb locus of each of four independently derived BALB/c plasmacytoid lymphosarcoma (ABPL9s) is due to the insertion of a defective Moloney murine leukemia virus (M-MuLV) into a 1.5-kilobase-pair stretch of cellular DNA at the 59 end of the v-myb-related sequences. This retroviral insertion is associated with abnormal transcription of myb sequences and probably represents a step in the neoplastic transformation of ABPL cells.

The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage lambda gtWES.lambda B was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments showed that subgenomic clones that lacked the 5' long terminal repeat and adjoining sequences (300 base pairs downstream of the repeat) were not biologically active. In contrast, subgenomic clones that lacked the 3' long terminal repeat and as much as 1.3 kilobase pairs of the A-MuLV cell-derived abl gene were as efficient as wild-type viral DNA in transformation. The A-MuLV-encoded polyprotein P120 and its associated protein kinase activity were detected in transformants obtained by transfection with Cla I, BamHI, and HindIII subgenomic clones. In contrast, individual transformants obtained with subgenomic Sal I clones expressed A-MuLV proteins ranging in size from 82,000 to 95,000 daltons. Each demonstrated an associated protein kinase activity. These results provide direct genetic evidence that only the proximal 40% of abl with its associated 5' helper viral sequences is required for fibroblast transformation.