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Chronic lymphocytic leukemia (CLL) is a B-cell malignancy with a mature phenotype. In spite of its relatively indolent nature, no radical cure is as yet available. CLL is not associated with either a unique cytogenetic or a molecular defect, which might have been a potential therapeutic target. Instead, several factors are involved in disease development, such as environmental signals which interact with genetic abnormalities to promote survival, proliferation and an immune surveillance escape. Among these, PI3-Kinase signal pathway alterations are nowadays considered to be clearly important. The TCL1 gene, an AKT co-activator, is the cause of a mature T-cell leukemia, as well as being highly expressed in all B-CLL. A TCL1 transgenic mouse which reproduces leukemia with a distinct immunophenotype and similar to the course of the human B-CLL was developed several years ago and is widely used by many groups. This is a review of the CLL biology arising from work of many independent investigators who have used TCL1 transgenic mouse model focusing on pathogenetic, microenviroment and therapeutic targets.

Chronic lymphocytic leukemia (CLL) patients with deletion of chromosome 17p, where the p53 gene is located, often develop more aggressive disease with poor clinical outcomes. To investigate the underlying mechanisms for the highly malignant phenotype of 17p- CLL and to facilitate in vivo evaluation of potential drugs against CLL with p53 deletion, we have generated a mouse model with TCL1-Tg:p53(-/-) genotype. These mice develop B-cell leukemia at an early age with an early appearance of CD5+ / IgM+ B cells in the peritoneal cavity and spleen, and exhibit an aggressive path of disease development and drug resistance phenotype similar to human CLL with 17p deletion. The TCL1-Tg:p53(-/-) leukemia cells exhibit higher survival capacity and are more drug resistant than the leukemia cells from TCL1-Tg:p53wt mice. Analysis of microRNA expression reveals that p53 deletion resulted in a decrease of miR-15a and miR-16-1, leading to an elevated expression of Mcl-1. Primary leukemia cells from CLL patients with 17p deletion also show a decrease in miR-15a/miR-16-1 and an increase in Mcl-1. Our study suggests that the p53/miR15a/16-1/Mcl-1 axis may be an important pathway that regulates Mcl-1 expression and contributes to drug resistance and aggressive phenotype in CLL cells with loss of p53.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia. Deregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B cells causes a CD5-positive leukemia similar to aggressive human B-CLLs. We recently reported that levels of TCL1 expression in B-CLL are regulated by miR-29 and miR-181 that target 3' UTR of TCL1. To determine whether treatment with microRNAs targeting TCL1 can inhibit B-CLL in mice, we generated TCL1 transgenic mice using a construct containing the 3' and 5' UTRs of TCL1 under B-cell-specific Emicro promoter (Emicro-TCL1FL). At the age of 16-20 months, these mice showed B-CLL-like disease. Immunophenotyping revealed accumulation of CD5+CD23+B220+ population in spleens and lymph nodes. Our results show that CD5+CD23+ B-cell populations from Emicro-TCL1FL mice actively proliferate and show significantly increased levels of phospho-Akt. Emicro-TCL1FL mice showed immunological abnormalities similar to human B-CLL, including hypoimmunoglobulinemia, abnormal levels of cytokines and impaired immune response. These findings revealed biochemical and immunological similarities between Tcl1-driven B-CLL in mice and human B-CLL.

Overexpression of the TCL1 gene family plays a role in the onset of T-cell leukemias in mice and in humans. The Tcl1 gene is tightly regulated during early embryogenesis in which it participates in embryonic stem (ES)-cells proliferation and during lymphoid differentiation. Here, we provide evidences that Tcl1 is also important in mouse hair follicle (HF) and skin homeostasis. We found that Tcl1(-/-) adult mice exhibit hair loss, leading to alopecia with extensive skin lesions. By analysing Tcl1 expression in the wild-type (wt) skin through different stages of hair differentiation, we observe high levels in the secondary hair germ (HG) cells and hair bulges, during early anagen and catagen-telogen transition phases. The loss of Tcl1 does not result in apparent skin morphological defects during embryonic development and at birth, but its absence causes a reduction of proliferation in anagen HFs. Importantly, we show the that absence of Tcl1 induces a significant loss of the stem-cell marker CD34 (but not alpha6-integrin) expression in the bulge cells, which is necessary to maintain stem-cell characteristics. Therefore, our findings indicate that Tcl1 gene(s) might have important roles in hair formation, by its involvement in cycling and self-renewal of transient amplifying (TA) and stem-cell (SC) populations.

T-cell chronic lymphocytic/prolymphocytic leukemia (T-CLL/T-PLL) is a lymphoproliferative disease derived from immunocompetent post-thymic T cells. Activation (initiation of expression) of the TCL1 locus at chromosome 14q32.1 appears to be the causal event in the pathogenesis of these mature T-cell leukemias. This activation occurs as a result of translocations or inversions that cause rearrangement of the TCL1 (T-cell leukemia/lymphoma 1) locus with regulatory elements of T-cell receptor genes. To describe the molecular events that take part in the leukemogenesis of mature T-cell leukemias, we reviewed the literature and our own data on the molecular basis of mature T-cell leukemia. This data search revealed that 4 genes have been identified at the TCL1 locus: TCL1, TCL1b, TNG1, and TNG2. The expression of these genes is substantially increased following rearrangements involving 14q32.1. Functional analysis of the Tcl1 protein revealed its involvement in an Akt (protein kinase B) prosurvival pathway through its interaction with the Akt kinase, which promotes translocation of Akt to the nucleus and increases Akt's enzymatic activity. The available data provide important insights into the molecular mechanisms of T-cell leukemogenesis that may lead to the development of new drugs for treatment of mature T-cell leukemia.

Tcl1 and Mtcp1, members of the Tcl1 family, are implicated in T-cell prolymphocytic leukemia. The crystal structure of a dimer of murine Tcl1 has been determined at 2.5 A resolution with an R factor of 0.225. Murine Tcl1, human Tcl1 and Mtcp1 share very similar subunit structures, with RMS differences of 0.6 and 1.4 A for C(alpha) atoms, respectively, while the sequences share 50 and 36% identity, respectively. These structures fold into an eight-stranded beta-barrel of unique topology and high internal symmetry of 1.1-1.3 A for the two halves of human and murine Tcl1 and 1.7 A for Mtcp1, despite the low 12-13% sequence identity. The molecular surfaces of all three structures showed a common planar region which is likely to be involved in protein-protein interactions.

The TCL1 locus on human chromosome 14q32.1 is activated in T-cell leukemias by translocations and inversions that juxtapose it to regulatory elements of T-cell receptor genes. We isolated and characterized four genes at this locus, TCL1 and TCL1b (T-cell leukemia/lymphoma 1 and 1b), and TNG1 and TNG2 (TCL neighboring genes 1 and 2) all of which are overexpressed following rearrangements involving 14q32.1. TCL1 and TCL1b show 60% similarity and are represented in the mouse by a cluster of six homologous genes. In humans TCL1 and TCL1b show similar expression patterns: They are expressed mainly in CD4-/CD8- immature T-cells, pre B-cells and virgin B-cells. Expression decreases significantly at more mature stages of B-cell development. Activation of TCL1 and/or TCL1b in mature T-cells causes T-cell leukemia in humans. The oncogenic nature of TCL1 was confirmed by the analysis of a transgenic mouse model. Functional analysis of Tcl1 revealed its involvement in a PI3-kinase dependent Akt (PKB) pro-survival pathway through its interaction with the Akt kinase which increases Akt's enzymatic activity and promotes translocation of Akt to the nucleus.

The immediate early gene NUR77 (also called NGFI-B) is required for T cell antigen receptor-mediated cell death and is induced to very high levels in immature thymocytes and T cell hybridomas undergoing apoptosis. The Akt (PKB) kinase is a key player in transduction of anti-apoptotic and proliferative signals in T cells. Because Nur77 has a putative Akt phosphorylation site at Ser-350, and phosphorylation of this residue is critical for the transactivation activity of Nur77, we investigated whether Akt regulates Nur77. Coimmunoprecipitation experiments showed the detection of Nur77 in Akt immune complexes, suggesting that Nur77 and Akt physically interact. We further show that Akt specifically phosphorylates Ser-350 of the Nur77 protein within its DNA-binding domain in vitro and in vivo in 293 and NIH 3T3 cells. Because phosphorylation of Ser-350 of Nur77 is critical for its function as a transcription factor, we examined the effect of Akt on this function. By using luciferase assay experiments, we showed that phosphorylation of Nur77 by Akt decreased the transcriptional activity of Nur77 by 50--85%. Thus, we show that Akt interacts with Nur77 and inactivates Nur77 by phosphorylation at Ser-350 in a phosphatidylinositol 3-kinase-dependent manner, connecting the phosphatidylinositol 3-kinase-dependent Akt pathway and a nuclear receptor pathway.

Objective: TCL1, MTCP1 and TCL1b are three members of a new family of oncogenes that are expressed in T cell leukemias of ataxia telangiectasia patients (T-PLL, T-CLL). TCL1 is located at 14q32.1 and activated by juxtaposition to the alpha/delta-locus at 14q11 or beta-locus at 7q35 of the T cell receptor during the reciprocal translocations t(14;14)(q11;q32), t(7;14)(q35;q32), or inversion inv(14)(q11;q32). TCL1 encodes a predominantly cytoplasmic protein of 114 aa (14 kD) of unknown function. Recent studies suggest that TCL1 promotes cell survival rather than stimulating cell proliferation, as previously proposed., Methods: In an attempt to clarify the contexts in which TCL1 is expressed, we investigated TCL1 expression in 114 lymphoma and leukemia patients by Northern blot, RT-PCR and immunohistochemistry., Results: TCL1 expression is restricted to lymphoid cells, and is found in neoplastic (T and B cell neoplasms, and Hodgkin's disease) and nonneoplastic proliferations (reactive lesions). Out of 114 cases, 18 neoplasms of myeloid and 4 cases of epithelial origin were TCL1-negative. In lesions of the lymphoid system, both low- and high-grade lymphomas were found to express TCL1., Conclusions: We propose that TCL1 expression especially in high-grade B cell non-Hodgkin's lymphomas might interfere with B cell differentiation and promote the transition from low- to high-grade lymphoma., (Copyright 2001 S. Karger AG, Basel)

Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb(-) SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1-E12-Lmo2 and tal-1-E12-Lmo2-Ldb1 complexes but not that of a tal-1-E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1-E12-Lmo2-Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.

Chromosomal rearrangements observed in T-cell prolymphocytic leukemia involve the translocation of one T-cell receptor gene to either chromosome 14q32 or Xq28, deregulating the expression of cellular protooncogenes of unknown function, such as TCL1 or its homologue, MTCP1. In the human hematopoietic system, TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells causes mature T-cell proliferation in transgenic mice. In this study, using a newly generated monoclonal antibody against recombinant TCL1 protein, we extended our analysis mainly by immunohistochemistry and also by fluorescence-activated cell sorting and Western blot to a large tumor lymphoma data bank including 194 cases of lymphoproliferative disorders of B- and T-cell origin as well as reactive lymphoid tissues. The results obtained show that in reactive lymphoid tissues, TCL1 is strongly expressed by a subset of mantle zone B lymphocytes and is expressed to a lesser extent by follicle center cells and by scattered interfollicular small lymphocytes. In B-cell neoplasia, TCL1 was expressed in the majority of the cases, including lymphoblastic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (60%), and primary cutaneous B cell lymphoma (55%). TCL1 expression was observed in both the cytoplasmic and nuclear compartments, as confirmed by Western blot analysis. Conversely, TCL1 was not expressed in Hodgkin/Reed-Sternberg cells, multiple myelomas, marginal zone B-cell lymphomas, CD30+ anaplastic large cell lymphoma, lymphoblastic T-cell lymphoma, peripheral T-cell lymphoma, and mycosis fungoides. These data indicate that TCL1 is expressed in more differentiated B cells, under both reactive and neoplastic conditions, from antigen committed B cells and in germinal center B cells and is down-regulated in the latest stage of B-cell differentiation.

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination., (Copyright 2000 Academic Press.)

The TCL1 oncogene at 14q32.1 is involved in the development of human mature T-cell leukemia. The mechanism of action of Tcl1 is unknown. Because the virus containing the v-akt oncogene causes T-cell lymphoma in mice and Akt is a key player in transduction of antiapoptotic and proliferative signals in T-cells, we investigated whether Akt and Tcl1 function in the same pathway. Coimmunoprecipitation experiments showed that endogenous Akt1 and Tcl1 physically interact in the T-cell leukemia cell line SupT11; both proteins also interact when cotransfected into 293 cells. Using several AKT1 constructs in cotransfection experiments, we determined that this interaction occurs through the pleckstrin homology domain of the Akt1 protein. We further demonstrated that, in 293 cells transfected with TCL1, the endogenous Akt1 bound to Tcl1 is 5-10 times more active compared with Akt1 not bound to Tcl1. The intracellular localization of Tcl1 and Akt1 in mouse fibroblasts was investigated by immunofluorescence. When transfected alone, Akt1 was found only in cytoplasm whereas Tcl1 was localized in the cytoplasm and in the nucleus. Interestingly, Akt1 was also found in the nucleus when AKT1 was cotransfected with TCL1, suggesting that Tcl1 promotes the transport of Akt1 to the nucleus. These findings were supported by the intracellular localization of Akt1 or Tcl1 when Tcl1 or Akt1, respectively, were confined to the specific cellular compartments. Thus, we demonstrate that Tcl1 is a cofactor of Akt1 that enhances Akt1 kinase activity and promotes its nuclear transport.

TCL1 and TCL1b genes on human chromosome 14q23.1 are activated in T cell leukemias by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T cell receptor genes. In this report we present the cloning, mapping, and expression analysis of the human and murine TCL1/Tcl1 locus. In addition to TCL1 and TCL1b, the human locus contains two additional genes, TCL1-neighboring genes (TNG) 1 and 2, encoding proteins of 141 and 110 aa, respectively. Both genes show no homology to any known genes, but their expression profiles are very similar to those of TCL1 and TCL1b. TNG1 and TNG2 also are activated in T cell leukemias with rearrangements at 14q32.1. To aid in the development of a mouse model we also have characterized the murine Tcl1 locus and found five genes homologous to human TCL1b. Tcl1b1-Tcl1b5 proteins range from 117 to 123 aa and are 65-80% similar, but they show only a 30-40% similarity to human TCL1b. All five mouse Tcl1b and murine Tcl1 mRNAs are abundant in mouse oocytes and two-cell embryos but rare in various adult tissues and lymphoid cell lines. These data suggest a similar or complementary function of these proteins in early embryogenesis.

The TCL1 oncogene on human chromosome 14q32.1 is involved in the development of T cell leukemia in humans. Its expression in these leukemias is activated by chromosomal translocations and inversions at 14q32.1. Here we report the isolation and characterization of a new member of the TCL1 gene family, TCL1b, located approximately 16 kb centromeric of TCL1. The 1.2-kb TCL1b cDNA encodes a 14-kDa protein of 128 aa and shows 60% similarity to Tcl1. Expression profiles of TCL1 and TCL1b genes are very similar: both genes are expressed at very low levels in normal bone marrow and peripheral lymphocytes but are activated in T cell leukemia by rearrangements of the 14q32.1 region. Thus, translocations and inversions at 14q32. 1 in T cell malignancies involve two oncogenes.

The TCL1 oncogene on human chromosome 14q32.1 is involved in the development of T cell leukemia in humans. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. The TCL1 oncogene is activated in these leukemias by juxtaposition to the alpha or beta locus of the T cell receptor, caused by chromosomal translocations t(14:14)(q11:q32), t(7:14)(q35:q32), or by inversions inv(14)(q11:q32). To show that transcriptional alteration of TCL1 is causally involved in the generation of T cell neoplasia we have generated transgenic mice that carry the TCL1 gene under the transcriptional control of the p56(lck) promoter element. The lck-TCL1 transgenic mice developed mature T cell leukemias after a long latency period. Younger mice presented preleukemic T cell expansions expressing TCL1, and leukemias developed only at an older age. The phenotype of the murine leukemias is CD4-CD8+, in contrast to human leukemias, which are predominantly CD4+CD8-. These studies demonstrate that transcriptional activation of the TCL1 protooncogene can cause malignant transformation of T lymphocytes, indicating the role of TCL1 in the initiation of malignant transformation in T prolymphocytic leukemias and T chronic lymphocytic leukemias.

The TCL-1 gene which is located on chromosome 14 plays a major role in human hematopoeitic malignancies and encodes a 14-kDa protein whose function has not been determined. The TCL-1 gene is expressed in pre-B cells, in immature thymocytes, and at low levels in activated T cells but not in peripheral mature B cells and in normal cells. The TCL-1 protein is similar in its primary structure to a protein encoded by the mature T cell proliferation gene (MTCP-1). The MTCP-1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T cell proliferative diseases. The TCL-1 and MTCP-1 genes appear to be members of a family of genes involved in lymphoid proliferation and T cell malignancies. Our laboratory has undertaken the study of the TCL-1 and MTCP-1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, both purified TCL-1 and MTCP-1 proteins in forms with and without a six His tag sequence. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymeric-based reverse-phase column. The MTCP-1 recombinant proteins display greater solubility, do not form disulfide linked dimers or oligomers, and elute at a lower isopropanol concentration than the corresponding TCL-1 proteins. The purified recombinant TCL-1 and MTCP-1 proteins have been characterized by N-terminal sequence analysis, time of flight mass spectrometry, and circular dichroism spectroscopy. Initial results have indicated that the MTCP-1 protein with the His tag removed is suitable for both NMR and X-ray crystallographic methods of structure determination.

Among mature postthymic T-cell leukemias, adult T-cell leukemia (ATL) has characteristic clinicopathological entities. The association with the human T-cell leukemia/lymphotropic virus type I is one of the distinctive etiopathogenetic features of this disease. However, unlike other acute transforming retroviruses, the human T-cell leukemia/lymphotropic virus type I lacks an oncogene within its genome. Other human postthymic leukemias, such as T-prolymphocytic leukemias, involve mostly the CD4 cellular subset and share many similarities to ATLs (aggressive course, cutaneous involvement, CD4+, CD29+, CD45RA- phenotype, and alpha-naphthyl-acetate esterase positivity). A chromosomal rearrangement at 14q32.1, involved in translocations or inversions with either the alpha/delta locus [t(14;14)(q11;q32.1), inv14(q11;q32.1)], or the beta-chain locus of the T-cell receptor [t(7;14)(q35;q32.1)] is found. These rearrangements disregulate a gene, TCL1, located at the 14q32.1 region, that we show is physiologically expressed in CD4/CD8 double-negative thymocyte cells, but not in more differentiated CD4+ and CD8+ subpopulations. Here, using molecular and immunocytochemical analysis, we report that TCL1 is also overexpressed in 10 of 10 ATL specimens, indicating that this gene may play an important role in the pathogenesis of this disease.

In human leukemias and lymphomas nonrandom chromosomal rearrangements cause changes in cell growth and/or survival in such a way as to promote malignancy. The detailed study of the biochemical and genetic pathways altered in human cancer requires the identification or development of models to allow the study and manipulation of cancer gene function. Recently, the breakpoint gene TCL1, involved in chromosome translocations observed mostly in mature T-cell proliferations and chronic lymphocytic leukemias (CLL), was isolated and characterized, and showed to be part of a new gene family of proteins involved in these tumors. The murine Tcl1 gene, is similar in sequence to the murine and human MTCP1 gene also involved in T cell leukemias. The murine Tcl1 gene was shown to reside on mouse chromosome 12 in a region syntenic to human chromosome 14. Furthermore, we show that the murine Tcl1 gene is expressed early in mouse embryonic development and demonstrates expression in fetal hematopoietic organs as well as in immature T and B cells. Characterization of the murine Tcl1 gene will help in developing a mouse model of CLL and would provide the best opportunity to study and decipher the role of TCL1 in malignant transformation.

The TAL-1 gene specifies a basic helix-loop-helix domain (bHLH) transcription factor, which heterodimerizes with E2A gene family proteins. tal-1 protein is abnormally expressed in the majority of T-cell acute lymphoblastic leukemias (T-ALLs). tal-1 is expressed and plays a significant role in normal erythropoietic differentiation and maturation, while its expression in early myeloid differentiation is abruptly shut off at the level of late progenitors/early differentiated precursors (G. L. Condorelli, L. Vitelli, M. Valtieri, I. Marta, E. Montesoro, V. Lulli, R. Baer, and C. Peschle, Blood 86:164-175, 1995). We show that in late myeloid progenitors (the phenotypically normal murine 32D cell line) and early leukemic precursors (the human HL-60 promyelocytic leukemia cell line) ectopic tal-1 expression induces (i) a proliferative effect under suboptimal culture conditions (i.e., low growth factor and serum concentrations respectively), via an antiapoptotic effect in 32D cells or increased DNA synthesis in HL-60 cells, and (ii) a total or marked inhibitory effect on differentiation, respectively, on granulocyte colony-stimulating factor-induced granulopoiesis in 32D cells or retinoic acid- and vitamin D3-induced granulo- and monocytopoiesis in HL-60 cells. Furthermore, experiments with 32D temperature-sensitive p53 cells indicate that aberrant tal-1 expression at the permissive temperature does not exert a proliferative effect but causes p53-mediated apoptosis, i.e., the tal-1 proliferative effect depends on the integrity of the cell cycle checkpoints of the host cell, as observed for c-myc and other oncogenes. tal-1 mutant experiments indicate that ectopic tal-1 effects are mediated by both the DNA-binding and the heterodimerization domains, while the N-terminally truncated tal-1 variant (M3) expressed in T-ALL malignant cells mimics the effects of the wild-type protein. Altogether, our results (i) indicate proliferative and antidifferentiative effects of ectopic tal-1 expression, (ii) shed light on the underlying mechanisms (i.e., requirement for the integrity of the tal-1 bHLH domain and cell cycle checkpoints in the host cell, particularly p53), and (iii) provide new experimental models to further investigate these mechanisms.

Conventional chromosome analysis (CCA) and fluorescent in situ hybridization (FISH) studies, using a 390-kb yeast artificial chromosome probe spanning the area of multiple breakpoints of the BCL1 locus at 11q13, were performed on 57 patients fulfilling the French-American-British criteria for the diagnosis of atypical B-cell chronic lymphocytic leukemia (CLL). To better define the incidence of 13q deletions and trisomy 12, FISH analysis was also performed using a cosmid probe that recognized a DNA sequence between the Rb gene and the D13S25 locus at band 13q14 and a chromosome 12-specific pericentromeric probe. All patients were characterized by cytoimmunological and hematological studies. Fourteen cases displayed three fluorescent signals in 41-98% interphase cells when hybridized to the BCL1 yeast artificial chromosome probe, documenting the presence of BCL1 translocation (BCL1-positive cases). The presence of t(11;14)(q13;q32) was ascertained in 12 cases using CCA and by dual color interphase FISH using the BCLI probe and a 14q telomere probe in 2 karyotypically normal cases. The remaining 43 cases had two signals in more than 95% interphase cells (BCL1-negative) and did not have the t(11;14) at CCA. Although 13q14 deletions were seen by means of CCA in only 5 of 14 BCL1-positive cases, hemizygous or homozygous deletions at band 13q14 were detected by FISH in 11 of 14 BCL1-positive cases, as compared with 17 of 43 BCL1-negative cases (P = 0.01). A subclone with trisomy 12 in addition to BCL1 translocation and del(13q14) was present in four BCL1-positive cases. We arrived at the following conclusions: (a) FISH with this BCL1 YAC probe is an efficient method for the detection of the t(11;14) and of the corresponding involvement of the BCL1 locus in this lymphoproliferative disorder; (b) the majority of BCL1-positive atypical CLLs by French-American-British criteria may carry 13q14 deletions; (c) the recognition of this cytogenetic subset of atypical CLL, sharing some immunological and cytogenetic features with mantle cell lymphoma, may be important, because these patients usually present isolated peripheral blood and marrow lymphocytosis, with or without mild to moderate spleen involvement, and may require early cytotoxic treatment.

The TAL-1 gene specifies for a basic domain-helix-loop-helix protein, which is involved in the control of normal hematopoiesis. In human pathology, the TAL-1 gene product is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range; however, it has not been established whether the expression has a causal role in oncogenesis. In this report, we describe the phenotype of mouse transgenic lines obtained by inducing tal-1 protein expression in lymphoid tissues using the LCK promoter. The survival rate of tal-1 transgenic animals was much lower as compared with control mice. Histopathological analysis revealed lymphomas of T-cell type, often comprising a minor B-cell component. Some mice showed marked splenic lymphocyte depletion. Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis. To further unravel the tal-1 oncogenic potential, a strain of tal-1 transgenic mice was crossbred with p53-/- mice; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice, and histopathological analysis revealed exclusively T-cell lymphomas. These data indicate that TAL-1, expressed in T cells, is per se a potent oncogene, which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias.

Treatment of prostate cancer cell lines expressing bcl-2 with taxol induces bcl-2 phosphorylation and programmed cell death, whereas treatment of bcl-2-negative prostate cancer cells with taxol does not induce apoptosis. bcl-2 phosphorylation seems to inhibit its binding to bax since less bax was observed in immunocomplex with bcl-2 in taxol-treated cancer cells. These findings support the use of the anticancer drug taxol for the treatment of bcl-2-positive prostate cancers and other bcl-2-positive malignancies, such as follicular lymphoma.

The TCL1 oncogene on human chromosome 14q32.1 is involved in chromosome translocations [t(14;14)(q11;q32.1) and t(7;14)(q35;q32.1)] and inversions [inv14(q11;q32.1)] with TCR alpha/beta loci in T-cell leukemias, such as T-prolymphocytic (T-PLL). It is also involved in T-acute and -chronic leukemias arising in cases of ataxia-telangiectasia (AT), an immunodeficiency syndrome. Similar chromosomal rearrangements occur also in the clonally expanded T cells in AT patients before the appearance of the overt leukemia. We have analyzed the expression of TCL1 mRNA and protein in peripheral blood lymphocytes (PBLs) from four AT cases and from healthy controls. We found that the TCL1 gene was overexpressed in the PBLs of an AT patient with a large clonal T-cell population exhibiting the t(14;14) translocation but not in the lymphocytes of the other cases. Fluorescence in situ hybridization of the TCL1 genomic locus to lymphocyte metaphases from the AT patient with the T-cell clonal expansion showed that the breakpoint of the t(14;14) translocation lies within the TCL1 locus and is accompanied by an inverted duplication of the distal part of chromosome 14. These data indicate that TCL1 is activated in preleukemic clonal cells as a consequence of chromosome translocation involving sequences from the TCR locus at 14q11. Deregulation of TCL1 is the first event in the initiation of malignancy in these types of leukemias and represents a potential tool for clinical evaluation.

The antiapoptosis potential of Bcl-2 protein is well established, but the mechanism of Bcl-2 action is still poorly understood. Using the phosphatase inhibitor okadaic acid or the chemotherapeutic drug taxol, we found that Bcl-2 was phosphorylated in lymphoid cells. Phospho amino acid analysis revealed that Bcl-2 was phosphorylated on serine. Under similar conditions, okadaic acid or taxol treatment led to the induction of apoptosis in these cells. Thus, phosphorylation of Bcl-2 seems to inhibit its ability to interfere with apoptosis. In addition, phosphorylated Bcl-2 can no longer prevent lipid peroxidation as required to protect cells from apoptosis.

The TCL1 locus on chromosome 14q32.1 is frequently involved in chromosomal translocations and inversions with one of the T-cell receptor loci in human T-cell leukemias and lymphomas. The chromosome 14 region translocated or rearranged involves approximately 350 kb of DNA at chromosome band 14q32.1. Within this region we have identified a gene coding for a 1.3-kb transcript, expressed only in restricted subsets of cells within the lymphoid lineage and expressed at high levels in leukemic cells carrying a t(14;14)(q11;q32) chromosome translocation or a inv(14)(q11;q32) chromosome inversion. The cognate cDNA sequence reveals an open reading frame of 342 nt encoding a protein of 14 kDa. The TCL1 gene sequence, which, to our knowledge, shows no sequence homology with other human genes, is preferentially expressed early in T- and B-lymphocyte differentiation.

The TCL-1 gene maps at chromosome 14q32.1 and is activated in T cell leukemias and lymphomas by either chromosome translocations or inversions that juxtapose the TCL-1 gene to the alpha/delta or the beta locus of the T cell receptor. The open reading frame of the TCL-1 gene, coding for a protein of 114 amino acids, was expressed in bacteria and antisera were raised against it. The antibodies recognized the predicted TCL-1 M(r) 14,000 protein product in cells expressing TCL-1 mRNA. Cell fractionation experiments indicated that the TCL-1 protein is present in the microsomal fraction. These results were confirmed by confocal microscopy. The TCL-1 protein has considerable sequence similarities to the product of the MTCP-1 gene on chromosome Xq28, which is involved in T cell lympho-proliferative diseases. Thus, TCL-1 may represent a member of a novel family of genes involved in lymphoid proliferation and/or survival and in T cell malignancies.

The oncogene product bcl-2 functions as a repressor of programmed cell death and is a 26-kDa protein with a single predicted transmembrane segment located at the carboxyl terminus. The bcl-2 protein seems to function in different subcellular compartments, as evidenced by several biochemical and ultrastructural studies. The present study was performed to purify bcl-2 protein in significant quantities necessary for structural and functional studies. For this purpose, the bcl-2 gene was over-expressed in either baculovirus system or lymphocytes. Initially, attempts were undertaken to purify bcl-2 protein using conventional methods such as ion exchange or gel filtration chromatography. During these purification attempts we determined that bcl-2 protein is highly hydrophobic and prone to aggregation as might be expected for an integral membrane protein. By ion exchange and gel filtration chromatography, this protein could be partially purified. In order to purify bcl-2 to apparent homogeneity and avoid the aggregation problem, we prepared immunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequence of human bcl-2. The antibody was either coupled to CNBr-activated Sepharose 4B or cross-linked into protein A-Sepharose by dimethylpimelimidate dihydrochloride. Cellular extract equivalent to 10(8) bcl-2-overexpressing insect cells or lymphocytes was applied to immunoaffinity columns. Approximately 500 micrograms purified bcl-2 protein could be recovered as estimated by silver staining and immunoblotting. Furthermore, purified bcl-2 protein was electroporated into Pre-B lymphocytes which do not express this protein in sufficient quantity to delay the onset of glucocorticoid-induced apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The antiapoptosis potential of bcl-2 has now been well established. But the biochemical mechanism of bcl-2 action is still poorly understood. Using the phosphatase inhibitor okadaic acid (OA) or chemotherapeutic agents such as Taxol and 5'-fluorouracil, we found that bcl-2 can be phosphorylated. Since OA or Taxol treatment leads to apoptosis, it seems that phosphorylation of bcl-2 leads to its inactivation. Exposure of several lymphoid cell lines expressing differential amounts of bcl-2 protein to OA resulted in apoptosis of the cells and hyperphosphorylation of bcl-2. Interestingly, the lymphoblastoid cell lines that did not phosphorylate bcl-2 following OA exposure did not undergo apoptosis. Moreover, pro-B cells isolated from patients with acute lymphoblastic leukemias exhibited endogenous phosphorylated forms of bcl-2 and a large number of apoptotic cells, even without OA treatment. Treatment with the phosphatase inhibitor or with chemotherapeutic agents (Taxol, 5'-fluorouracil) led to severe apoptosis of these cells, along with hyperphosphorylation of bcl-2. Phosphoamino acid analysis reveals that bcl-2 is phosphorylated at a serine residue. In summary, our investigation indicates that the phosphorylation pathway involving bcl-2 can be the determinant of cell death in lymphocytes.

Evidence from cytogenetics, multipoint linkage analyses of familial melanoma, and loss of heterozygosity studies of familial and sporadic melanomas support localization of a melanoma susceptibility or tumor suppressor gene at chromosomal region 9p21-23. Recently, the inhibitor of cyclin-dependent kinase 4 (CDK4I; also known as p16INK4, multiple tumor suppressor 1, or CDKN2 gene) has been mapped to 9p21 and shown to be mutated or deleted in a large fraction of cell lines derived from many tumor types, including melanoma, suggesting that this gene could be a melanoma suppressor gene. In order to test for somatic mutations in the CDK4I gene in tumors, DNAs from 30 surgically resected melanomas of both cutaneous and uveal origins were sequenced. No mutations were detected in the coding region of the CDK4I gene, while mutations or deletions were detected in 60% (9 of 15) of the cultured melanoma cell line DNAs. Among presumptive familial cases, nine of which were members of families with one or two other documented melanoma cases, no germline mutations were detected by sequence analysis. A deletion in the second exon of the CDK4I gene was found in one germline allele of a familial melanoma patient from a family with eight affected first degree relatives. These results not only support the suggestion that the CDK4I gene is a familial malignant melanoma gene, they also suggest the presence of another suppressor gene locus within 9p21 which is the target of loss of heterozygosity in sporadic melanomas.

The bcl-2 protein plays a central role in the regulation of programmed cell death in a variety of tissues and is pivotal to the survival of lymphocytes in vivo. The growth factors responsible for survival of normal lymphocytes are unknown but are likely to maintain viability in part through the regulation of bcl-2 expression. A subset of human natural killer (NK) cells (CD3-CD56bright) are unique among lymphocytes in their constitutive expression of c-kit, a tyrosine kinase cell surface receptor that binds c-kit ligand (KL). Alone, KL does not promote proliferation or further differentiation of CD56bright NK cells. We now report that, in the absence of serum or additional growth factors, KL prevents apoptosis of cultured CD56bright NK cells, as assessed by DNA fragmentation studies, and maintains viability, as measured by biologic responses (i.e., proliferation and cytotoxicity) to the subsequent addition of other cytokines. Furthermore, we demonstrate that KL induces CD56bright NK cells to express the bcl-2 protein. In the presence of anti-c-kit antibody, the tyrosine kinase inhibitor genistein, or bcl-2 antisense oligonucleotide, the protective effect of KL on the survival of CD56bright NK cells is dramatically reduced. These data demonstrate that the binding of KL to its tyrosine kinase receptor results in the upregulation of bcl-2, thereby preventing apoptosis in this subset of normal human lymphocytes. As soluble KL is plentiful in normal human serum, this survival mechanism may be operative for CD56bright NK cells in vivo.

bcl-2 and p53 gene products have been both linked to programmed cell death pathways. We have analyzed several human breast cancer cell lines for the expression of bcl-2 and p53. We found an inverse correlation between the expression of the two proteins. The result suggested that mutant p53 could substitute for bcl-2 function in breast cancer cells and that could also down-regulate bcl-2 expression. We found, indeed, that overexpression of a mutant p53 (mut 175) in MCF-7 cells could induce down-regulation of bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gene does not contain the negative regulatory element responsible for the down-regulation. If this mechanism will be proved also for the wild-type p53 allele, it may disclose a possible mechanism for p53-induced apoptosis: down-regulation of bcl-2.

To better understand the role of the BCL-3 locus at chromosome 17q22 in the pathogenesis and progression of leukemias and lymphomas, we examined its genomic configuration in 264 B-cell malignancies and its expression in a smaller subset. Cases studied included 39 chronic lymphocytic leukemias, 58 low-grade follicular lymphomas, 20 mantle cell lymphomas, 30 small noncleaved cell lymphomas, 25 acute lymphoblastic leukemias, 10 acquired immunodeficiency syndrome--related non-Hodgkin's lymphomas, and 44 diffuse mixed- or diffuse large-cell lymphomas. In addition, 38 aggressive lymphomas (transformed follicular lymphomas) derived from previously indolent follicular lymphomas were examined. Southern blot analysis showed BCL-3 locus rearrangement in 4 cases (1.5%), ie, in 3 transformed follicular lymphomas and in 1 indolent follicular lymphoma. All 4 also had BCL-2 rearrangements consistent with their follicular center cell origin. None of the BCL-3 rearranged cases showed MYC gene rearrangement, as reported for the original leukemia that led to the discovery of BCL-3. Pretransformation specimens of all three transformed follicular lymphomas showed the presence of the BCL-3 alteration before histologic progression. In 1 case, serial pretransformation biopsies showed that the BCL-3 rearrangement was acquired during the indolent follicular phase of the patient's disease. Thirty lymphomas, including 2 of the 4 with BCL-3 rearrangement, were also examined for BCL-3 message. All 30, including the 2 with BCL-3 rearrangements, expressed the normal 1.7-kb BCL-3 transcript, at approximately equivalent levels. The data indicate that, although BCL-3 locus alterations are found in only a small fraction of B-cell lymphoid malignancies, they occur primarily in a subset of follicular center cell lymphomas. Interestingly, these alterations appear to be acquired during the indolent (follicular) phase of the disease and they are maintained when histologic transformation takes place. The data also suggest that BCL-3 locus alterations do not result in gross changes of BCL-3 gene expression and do not necessarily involve the MYC gene. Although the preferential involvement of BCL-3 alterations in a small subset of follicular lymphomas that transform suggests a possible link between these abnormalities and progression, further studies are needed to ensure that these alterations are biologically relevant and not simply a manifestation of genomic instability.

Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virus-induced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32P(i) demonstrated that BCL2 is a phosphoprotein.

Tre is a recombinant gene isolated from NIH3T3 cells transfected with human Ewing's sarcoma DNA. It is composed of three major genetic elements derived, 5' to 3', from human chromosomes 5, 18 and 17. We report here on transcripts from the 3' domain of tre. The transcripts were cloned from a cDNA library of cytoplasmic poly(A)+ RNA from tre-transfected NIH3T3 tumor cells. The complete cDNA sequence, 8201 nucleotides, possessed an unusually long non-coding region and a translatable region with two open reading frames. In one cDNA clone, the presence of two insertions suggested the possibility of alternative splicing. The sequence mapped to the centromere-proximal region of 17q. Transfection-tumorigenicity assays with the open reading frames subcloned into expression vectors were positive for the reading frame adjacent to the 5' non-coding region and negative for the second, downstream, reading frame and the possible alternatively spliced versions of both reading frames. Analysis of the 786 amino acid sequence deduced from the 5' reading frame predicted a highly hydrophilic protein with two charge clusters suggesting nucleic acid-binding properties. When used as probe, the cloned sequence detected RNA transcripts in a wide variety of human cancer cells regardless of their lineage of origin from different tissues, but not in human cells from normal tissue.

To determine the role of BCL-2 in the glucocorticoid-induced apoptosis of lymphocytes, we analyzed the effect of glucocorticoid on two human pre-B-cell lines which express different levels of BCL-2. Glucocorticoid treatment of the 380 cell line which expresses high levels of BCL-2 resulted in inhibition of cellular proliferation without induction of apoptosis. On the other hand, glucocorticoid treatment of the 697 cell line which expresses lower levels of the BCL-2 resulted in both inhibition of cellular proliferation and apoptosis with characteristic internucleosomal DNA cleavage. The glucocorticoid-induced inhibition of cellular proliferation in both cell lines was also associated with repression of the c-myc mRNA expression. Taken together, our data suggest that BCL-2 blocks the glucocorticoid-induced apoptosis of the 380 pre-B-lymphocytes by extending their survival when the level of c-myc expression is repressed. Also by repressing the expression of c-myc, glucocorticoid causes apoptosis of the 697 pre-B-lymphocytes in the absence of high level of BCL-2 expression.

The MYB related loci, AMYB and BMYB, were localized to specific human chromosome regions by Southern blot analysis of their segregation patterns in a panel of rodent-human hybrid DNAs using radiolabeled AMYB and BMYB probes. The AMYB locus was present in hybrids retaining the chromosome region 8cen----8q22 and was absent in hybrids which had lost this chromosome region. The presence of the BMYB locus in rodent-human hybrids correlated with, and only with, chromosome region Xq13. Chromosomal in situ hybridization refined the localization of AMYB to region 8q22-23 and confirmed the localization of BMYB to region Xq13. Chromosome region 8q22 is involved in recurrent translocations in malignant lymphoma and in acute myeloid leukemia (AML-M2); therefore AMYB is a candidate for involvement in such translocations. A region on Xq13 is also involved in chromosomal abnormalities in acute myeloid leukemia and myelodysplasias.

The role of bcl-2 in the signal transduction pathway was assessed by studying the inositol phospholipid metabolism in fibroblasts transfected by this oncogene. The rate of accumulation of water soluble inositol phosphates in response to several growth factors was much higher in bcl-2 transfected NIH3T3 clones than in untransfected control. Moreover, bcl-2 transfected clones express elevated levels of phosphatidic acid, a phospholipid produced during receptor stimulated breakdown of phosphoinositides. Our findings suggest that the expression of bcl-2 in NIH3T3 fibroblasts leads to the coupling of growth factor receptors to stimulate inositol phosphate production, henceforth establishing its role in the growth factor receptor mediated signal transduction pathway.

Antisense oligodeoxynucleotides specific for sequences in mRNAs from the B-cell lymphoma/leukemia-2 (BCL2) gene were used to inhibit the growth in culture of a human leukemia cell line, 697. Normal phosphodiester (PO) and nuclease-resistant phosphorothioate (PS) oligodeoxynucleotides were compared with regard to specificity, potency, and kinetics. Both PO and PS antisense BCL2 oligodeoxynucleotides were specific inhibitors of cellular proliferation, since sense versions of these synthetic DNAs were inactive at similar concentrations. Specificity was further confirmed by quantitative immunofluorescence studies, showing that PO and PS antisense BCL2 oligodeoxynucleotides (when used at appropriate concentrations) reduced levels of BCL2 protein without influencing expression of HLA-DR and other control antigens. The onset of inhibition by PO oligodeoxynucleotides was faster, with reductions in cell numbers occurring within 1 day of addition to cultures, in contrast to phosphorothioates, which were ineffective until 3-4 days. Phosphorothioates were more potent that phosphodiesters, however, with half-maximal inhibition of leukemic cell growth occurring at concentrations 5-10 times lower. As expected from previous studies demonstrating the importance of BCL2 for regulating lymphoid cell survival, BCL2 antisense oligodeoxynucleotides also led to 697 leukemic cell death through sequence-specific mechanisms, with reductions in cellular viability generally lagging the inhibitory effects on cellular growth by about 2 days. Taken together, these data indicate that PO and PS oligodeoxynucleotides targeted against the human BCL2 protooncogene can be sequence-specific inhibitors of leukemic cell growth and survival.

The BCL2 (B cell lymphoma/leukemia-2) and C-HA-RAS oncogenes encode membrane-associated proteins of 26 and 21 kilodaltons, respectively. Although RAS proteins have long been known for their ability to bind and hydrolyze GTP, recent investigations suggest that BCL2 encodes a novel GTP-binding protein (S. Haldar, C. Beatty, Y. Tsujimoto, and C. M. Croce, Nature [London] 342:195-198, 1989). Cotransfection of BCL2 and HA-RAS oncogenes resulted in morphological transformation of early-passage rodent fibroblasts, rendering these cells tumorigenic in animals and enabling them to grow in semisolid medium. In contrast, cotransfection of BCL2 with oncogenes that encode nuclear proteins (E1A and C-MYC) did not produce malignant transformation, whereas HA-RAS did complement with these genes. These findings suggest that proteins encoded by oncogenes such as BCL2 and HA-RAS, although having similar subcellular locations and perhaps similar biochemical properties, can regulate distinct complementary pathways involved in cellular transformation.

The human homolog, c-ros, of the transforming gene, v-ros, of the avian sarcoma virus, UR2, has been isolated from a human genomic library. A single-copy fragment from the human c-ros genomic clone has been used to map the human c-ros homolog (ROS) to human chromosome region 6q16----6q22 by somatic cell hybrid analysis and chromosomal in situ hybridization. Thus, the c-ros gene joins the c-myb oncogene, which is distal to the c-ros gene on the long arm of human chromosome 6, as a candidate for involvement in chromosome 6q deletions and rearrangements seen in various malignancies.

The myb-ets-containing acute leukemia virus, E26, transforms myeloblasts and erythroblasts in culture and causes a mixed erythroid and myeloid leukemia in chicks. Genes (ets-1, ets-2, and erg) with variable relatedness to the v-ets oncogene of the E26 virus have been identified, cloned, and characterized in several species. Two new members (elk-1 and elk-2) of the ets oncogene superfamily have now been identified. Nucleotide sequence analysis of the elk-1 cDNA clone revealed that this gene encodes a 428-residue protein whose predicted amino acid sequence showed 82% similarity to the 3' region of v-ets. The elk or related sequences appear to be transcriptionally active in testis and lung. The elk cDNA probe detects two loci in the human genome, elk-1 and elk-2, which map to chromosome regions Xp11.2 and 14q32.3, respectively. These loci are near the translocation breakpoint seen in the t(X;18) (p11.2;q11.2), which is characteristic of synovial sarcoma, and the chromosome 14q32 breakpoints seen in ataxia telangiectasia and other T cell malignancies. This suggests the possibility that rearrangements of elk loci may be involved in pathogenesis of certain tumors.

The mouse PIM-1 gene is involved in the pathogenesis of virally-induced mouse lymphomas. We have cloned and analyzed the human homologue of the mouse PIM-1 gene to investigate its role in human lymphoma and leukemia. Overlapping cDNA clones from a K562 (human erythroleukemia cell line) library were isolated and sequenced. The deduced amino acid sequence showed significant homology to a number of the protein kinases but did not have a transmembrane region. Genomic clones from the 380 cell line (human B cell leukemia) were analyzed. The PIM-1 transcript was found to derive from 5 Kb of genomic DNA. Six exons and five introns were identified. The promoter region has no TATA or CAAT boxes, but did have multiple potential Sp1 binding sites (CCGCCC). Studies of expression of this gene using Northern blots of human cell lines showed it to be transcribed primarily in B lymphoid and myeloid cell lines. The characterization of the human PIM-1 gene will allow the definition of its role in hemopoietic malignancies and in hematolymphoid differentiation.

The mouse PIM-1 gene has been implicated in the evolution of retrovirus-associated mouse lymphomas. We have initiated a study of the human PIM-1 gene because of its potential importance as a human oncogene. We have isolated genomic and cDNA clones for this gene and characterized this locus in detail. The predicted PIM-1 protein is 313 amino acids in length. It has homology to a number of the protein kinases but does not have a transmembrane region. The amino acid corresponding to tyrosine-416 of pp60v-src is a tyrosine (position 198), which is consistent with the hypothesis that PIM-1 is a tyrosine kinase rather than a serine-threonine kinase. The PIM-1 gene was found to have six exons and five introns derived from 5 kb of genomic DNA. The site of transcription initiation was localized by S1 nuclease protection studies which indicated that the mature PIM-1 mRNA was approximately 2.7 kb in length. The promotor of this gene had no TATA or CAAT box but did have multiple GC boxes (CCGCCC) that might bind the Sp1 protein. The PIM-1 gene was expressed in myeloid and B lymphoid cell lines, but not in T lymphoid and nonhemopoietic lines. This initial characterization of PIM-1 will allow us to define its role in normal and malignant hematolymphoid differentiation.

A protein corresponding in size to the bcl-2 alpha protein obtained after in vitro transcription and in vitro translation in a rabbit reticulocyte lysate was specifically immunoprecipitated from a human B-cell extract using rabbit polyclonal antiserum raised against a beta-galactosidase/bcl-2 fusion protein. Peptide mapping with V8 protease confirmed the identity of this protein as human bcl-2 alpha. Subcellular fractionation of cellular protein followed by immunoprecipitation showed that the bcl-2 alpha protein is associated with the cell membrane.

We have analyzed the oncogene rearrangements involving BCL2 and MYC in the leukemia cells of a patient with an aggressive prolymphocytic leukemia that had an abnormal karyotype including a t(14;18) translocation and a chromosome 17q+. Molecular analysis showed that BCL2 was rearranged in the major breakpoint cluster region and had joined into the immunoglobulin heavy chain gene as in follicular lymphoma. Cloning and sequence analysis of the rearranged MYC gene revealed that MYC was truncated at the Pvu II site at the end of the first exon of MYC and had joined into the regulatory elements of a gene that we called BCL3 (B-cell leukemia/lymphoma 3). The BCL3 locus was mapped to chromosome 17 band q22. We found BCL3 transcribed as a message of 1.7 kilobases in many hematopoietic cell lines representing all hematopoietic lineages. In the patient's leukemia cells, the truncated MYC gene was highly expressed under the influence of BCL3 regulatory elements, leading to an aggressive B-cell leukemia that presumably had been derived from an indolent lymphoma carrying a rearranged BCL2 gene.

Little is known about the biochemical or functional nature of the proteins encoded by the bcl-2 gene, which undergoes chromosomal translocation in approximately 85% of follicular lymphoma, 20% of diffuse large cell lymphoma and 10% of chronic lymphocytic leukaemia of B cells. Translocation of bcl-2 sequences from chromosome 18 to the JH segment of the immunoglobulin gene at chromosome band 14q32 in B cells results in deregulated expression of this gene, causing high steady state levels of bcl-2 messenger RNA2. DNA sequence data indicate that bcl-2 encodes two proteins by virtue of alternative splicing, designated as Bcl-2 alpha and Bcl-2 beta, with relative molecular masses of 26,000 and 22,000 respectively. Cell fractionation experiments indicate that the bcl-2 alpha gene product is located at the inner surface of the cell membrane, suggesting a possible role in mitogenic signal transduction. We report here that Bcl-2 alpha has GTP-binding activity and a protein sequence that suggests it belongs to the small molecular weight GTP-binding protein (G protein) family.

The complete nucleotide sequence of human abl RNA containing exon Ia was determined. It spans 5598 nucleotides and codes for a protein of 1130 amino acids. The 3' untranslated region contains two short open reading frames and multiple ATTT(A) motifs characteristic of short lived mRNAs. Computer analysis of the abl protein predicts four domains distinct with regard to surface probability and chain flexibility. Nucleotide analysis of the abl segment within a bcr-abl cDNA cloned from the K562 cell line indicated no further alterations within the coding region. A bcr-abl construct containing this segment transformed, together with c-myc, RAT-1 cells and produced a highly active tyrosine kinase.