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Recent investigations of chromosomal aberrations in chronic lymphocytic leukemia (CLL) led to a better understanding of the molecular causes of CLL. Here we report a rearrangement between MAML2 (mastermind-like protein 2) and CXCR4 (specific receptor for CXC chemokine stromal cell-derived factor-1) in CLL cells of a patient with a t(2;11)(q22.1;q21) chromosomal translocation. The rearrangement between MAML2 and CXCR4, created by a t(2;11)(q22.1;q21) translocation, results in a new fusion gene in which a portion of CXCR4 is linked to the MAML2 gene. This fusion gene encodes for CXCR4/MAML2 protein chimera in which the N-terminal basic domain of MAML2 is replaced by the N-terminal domain of CXCR4., (© 2014 by The American Society of Hematology.)

The Leucine Zipper Tumor Suppressor 1 (LZTS1) is a tumor suppressor gene, located at chromosome 8p22, which is frequently altered in human cancer. In normal tissue, its ubiquitous expression regulates cell mitosis by the stabilization of microtubule networks. LZTS1-deficient mouse embryonic fibroblasts have been shown to have an accelerated mitotic progression, and a higher resistance to taxanes, microtubule-stabilizing drugs. We investigate the role of Lzts1 in paclitaxel-resistance in breast cancer cells. Downregulation of Lzts1 expression significantly decreases sensitivity to paclitaxel in vitro. We further analyzed Lzts1 expression by immunohistochemistry in 270 primary breast cancer samples and 16 normal breast specimens. Lzts1 was significantly downregulated in breast cancer samples and its deregulation was associated with a higher incidence of tumor recurrence, and to a worse overall survival. Moreover, Lzts1-negative tumors were associated with unfavorable outcome after taxanes-based therapy. Thus our data suggest that Lzts1 deregulation is involved in breast cancer and its immunohistochemical evaluation may serve as a prognostic factor for breast cancer therapy.

In sarcoma, the activity of NF-κB (nuclear factor κB) reduces the abundance of the microRNA (miRNA) miR-29. The tumor suppressor A20 [also known as TNFAIP3 (tumor necrosis factor-α-induced protein 3)] inhibits an upstream activator of NF-κB and is often mutated in lymphomas. In a panel of human sarcoma cell lines, we found that the activation of NF-κB was increased and, although the abundance of A20 protein and mRNA was decreased, the gene encoding A20 was rarely mutated. The 3' untranslated region (UTR) of A20 mRNA has conserved binding sites for both of the miRNAs miR-29 and miR-125. Whereas the expression of miR-125 was increased in human sarcoma tissue, that of miR-29 was decreased in most samples. Overexpression of miR-125 decreased the abundance of A20 mRNA, whereas reconstituting miR-29 in sarcoma cell lines increased the abundance of A20 mRNA and protein. By interacting directly with the RNA binding protein HuR (human antigen R; also known as ELAVL1), miR-29 prevented HuR from binding to the A20 3'UTR and recruiting the RNA degradation complex RISC (RNA-induced silencing complex), suggesting that miR-29 can act as a decoy for HuR, thus protecting A20 transcripts. Decreased miR-29 and A20 abundance in sarcomas correlated with increased activity of NF-κB and decreased expression of genes associated with differentiation. Together, the findings reveal a unique role of miR-29 and suggest that its absence may contribute to sarcoma tumorigenesis.

The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway.

The T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas.

DNA-damaging therapies represent a keystone in cancer treatment. Unfortunately, many tumors often relapse because of a group of cancer cells, which are resistant to conventional therapies. High-mobility group A (HMGA) proteins has a key role in cell transformation, and their overexpression is a common feature of human malignant neoplasias, representing a poor prognostic index often correlated to anti-cancer drug resistance. Our previous results demonstrated that HMGA1 is a substrate of ataxia-telangiectasia mutated (ATM), the main cellular sensor of genotoxic stress. Here we also report thatHMGA2, the other member of the HMGA family, is a novel substrate of ATM. Interestingly, we found that HMGA proteins positively regulate ATM gene expression. Moreover, induction of ATM kinase activity by DNA-damaging agents enhances HMGA-dependent transcriptional activation of ATM promoter, suggesting that ATM expression is modulated by a DNA-damage- and HMGA-dependent positive feedback loop. Finally, inhibition of HMGA expression in mouse embryonic fibroblasts and in cancer cells strongly reduces ATM protein levels, impairing the cellular DNA-damage response and enhancing the sensitivity to DNA-damaging agents. These findings indicate this novel HMGA-ATM pathway as a new potential target to improve the effectiveness of conventional anti-neoplastic treatments on the genotoxic-drug resistant cancer cells.

The high mobility group HMGA1 protein belongs to a family of architectural factors that play a role in chromosomal organisation and gene transcription regulation. HMGA1 overexpression represents a common feature of human malignant tumours and is causally associated with neoplastic transformation and metastatic progression. Recently, HMGA1 expression has been correlated with the presence of chromosomal rearrangements and suggested to promote genomic instability. Here, we report a novel interaction between HMGA1 protein and the ataxia-telangiectasia mutated (ATM) kinase, the major key player in the cellular response to DNA damage caused by several agents such as ionising radiation (IR). We identified an SQ motif on HMGA1, which is effectively phosphorylated by ATM in vitro and in vivo. Interestingly, confocal microscopy revealed that HMGA1 colocalises with the activated form of ATM (ATM S1981p). Moreover, HMGA1 ectopic expression decreases cell survival following exposure to IR as assessed by clonogenic survival in MCF-7 cells, further supporting the hypothesis that HMGA1 might act as a downstream target of the ATM pathway in response to DNA damage.

FEZ1/LZTS1 is a tumor suppressor gene that is frequently altered in human cancers of different histotypes. We have reported previously that LZTS1 is downregulated in high-grade bladder cancer and that its restoration suppresses tumorigenicity in urothelial carcinoma cells. To further investigate the role of LZTS1 in the development of bladder cancer, we utilized heterozygous and nullizygous Lzts1 mice in a chemically induced carcinogenesis model. Fifty-eight mice consisting of 25 Lzts1(+/+), 17 Lzts1(+/-) and 16 Lzts1(-/-) were treated with N-butyl-N-(4-hydroxybutil) nitrosamine (BBN). Results showed that there was a significant increase in neoplastic lesions in the Lzts1(+/-) (82.3%) and Lzts1(-/-) (93.8%) versus Lzts1(+/+) (8.0%) mice after BBN treatment. No difference in cancer incidence between Lzts1(+/-) and Lzts1(-/-) was observed. Collectively, these findings indicate that loss of one or both LZTS1 alleles hampers the normal defenses of urothelial cells against carcinogens, favoring bladder cancer development. Therefore, LZTS1 may become an excellent target for gene therapy in advanced bladder carcinoma.

p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The alpha isoforms of TAp63 and DeltaNp63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63alpha can directly inhibit its activity through an intramolecular interaction; similarly, DeltaNp63alpha can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63alpha isoforms is cleaved by activated caspases. Cleavage of DeltaNp63alpha relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63alpha results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

The Testin (TES) gene was previously identified as a putative human tumor suppressor gene at 7q31.2, a region that is frequently deleted in hematopoietic malignancies, as well as in epithelial tumors. To determine whether TES acts as a tumor suppressor in vivo, we generated a Tes knockout mouse and then used it in an established model of carcinogen-induced gastric cancer. In mice a zinc-deficient (ZD) diet enhances cellular proliferation in the forestomach and susceptibility to N-nitrosomethylbenzylamine (NMBA)-induced carcinogenesis. Five-week-old Tes wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were divided into four groups: mice fed a zinc-sufficient diet (ZS); mice fed a ZD diet; ZS fed plus NMBA-treated mice (ZS+NMBA), and ZD fed plus NMBA-treated mice (ZD+NMBA). After 4 weeks, the ZS+NMBA and ZD+NMBA groups were treated with three intragastric doses of NMBA. Animals were killed 8 weeks after NMBA administration: 25% of +/+ mice developed benign lesions; 88% of +/- showed multiple papillomas, atypical glandular metaplasia, and squamous cell carcinomasl; and 81% of -/- mice displayed very large papillomas, squamous cell carcinomas, and adenocarcinomas. A statistically significant difference in tumor incidence was found between +/- versus +/+ and -/- versus +/+ (P < 0.0001). These data suggest that Tes functions as a tumor suppressor gene in vivo.

HMGA1 (high-mobility-group A1) proteins are architectural transcription factors that are found overexpressed in embryogenesis and malignant tumours. We have shown previously that they have a role in lymphopoiesis, since the loss of HMGA1 expression leads to an impairment of T-cell development and to an increase in B-cell population. Since RAGs (recombination activating genes) are key regulators of lymphoid differentiation, in the present study we investigate whether RAG2 expression is dependent on HMGA1 activity. We show that RAG2 gene expression is up-regulated in Hmga1-/- ES (embryonic stem) cells and EBs (embryoid bodies) as well as in yolk sacs and fibroblasts from Hmga1-/- mice, suggesting that HMGA1 proteins control RAG2 gene expression both in vitro and in vivo. We show that the effect of HMGA1 on RAG2 expression is direct, identify the responsible region in the RAG2 promoter and demonstrate binding to the promoter in vivo using chromatin immunoprecipitation. Since RAG2 is necessary for lymphoid cell development, our results suggest a novel mechanism by which HMGA1 might regulate lymphoid differentiation.

The mixed-lineage leukemia (MLL1/ALL-1/HRX) histone methyltransferase is involved in the epigenetic maintenance of transcriptional memory and the pathogenesis of human leukemias. To understand its role in cell type specification, we determined the human genomic binding sites of MLL1. We found that MLL1 functions as a human equivalent of yeast Set1. Like Set1, MLL1 localizes with RNA polymerase II (Pol II) to the 5' end of actively transcribed genes, where histone H3 lysine 4 trimethylation occurs. Consistent with this global role in transcription, MLL1 also localizes to microRNA (miRNA) loci that are involved in leukemia and hematopoiesis. In contrast to the 5' proximal binding behavior at most protein-coding genes, MLL1 occupies an extensive domain within a transcriptionally active region of the HoxA cluster. The ability of MLL1 to serve as a start site-specific global transcriptional regulator and to participate in larger chromatin domains at the Hox genes reveals dual roles for MLL1 in maintenance of cellular identity.

The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

Chromosomal deletions are often observed in lung cancers suggesting that inactivation of tumor suppressor genes plays an important role in the development of this neoplasm. The region around chromosome 8p22 is a frequent and early target of these deletions and has therefore been investigated for the presence of candidate genes. The FEZ1/LZTS1 gene, located at 8p22, is inactivated in many cancers with 8p deletions, including prostate, esophageal, gastric, bladder, and breast cancer and the Fez1 protein has been shown to suppress growth of cancer cells and to regulate mitosis. To elucidate the role of FEZ1 in lung cancer, we have analyzed its expression by immunohistochemistry in 103 primary lung cancer specimens including 98 non-small cell lung cancers (57 adenocarcinomas, 32 squamous cell carcinomas, 7 large cell carcinomas, and 2 others) and five small cell carcinomas. Absence of Fez1 protein expression was observed in 27 cases (26%) and additional 43 cases (42%) showed strong reduction in immunoreactivity. There was a positive association between loss of FEZ1 expression and tumor grading (P = 0.0345) and a tendency toward a reduction in the mortality rate in subjects with strong FEZ1 expression. Overall, these data indicate an important role for FEZ1 in lung cancer and suggest the possibility that it may serve as a novel prognostic indicator.

The WWOX gene encodes a tumor suppressor WW domain-containing protein, Wwox. Alterations of WWOX have been demonstrated in multiple types of cancer, and introduction of Wwox into Wwox-negative tumor cells has resulted in tumor suppression and apoptosis. The Wwox protein contains two WW domains that typically bind proline-rich motifs and mediate protein-protein interactions. Recently, we have described functional cross-talk between the Wwox protein and the p53 homologue, p73. To further explore the biological function of Wwox, we investigated other interacting candidates. In this report, we demonstrate a physical and functional association between AP-2gamma transcription factor and the Wwox protein. AP-2gamma at 20q13.2 encodes a transcription factor and is frequently amplified in breast carcinoma. We show that Wwox binds to the PPPY motif of AP-2gamma via its first WW domain. Alterations of tyrosine 33 in the first WW domain of Wwox or the proline-rich motif in AP-2gamma dramatically reduce this interaction. In addition, our results demonstrate that Wwox expression triggers redistribution of nuclear AP-2gamma to the cytoplasm, hence suppressing its transactivating function. Our results suggest that Wwox tumor suppressor protein inhibits AP-2gamma oncogenic activity by sequestering it in the cytoplasm.

The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. Wwox protein contains two WW domains that are generally known to mediate protein-protein interaction. Here we show that Wwox physically interacts via its first WW domain with the p53 homolog, p73. The tyrosine kinase, Src, phosphorylates Wwox at tyrosine 33 in the first WW domain and enhances its binding to p73. Our results further demonstrate that Wwox expression triggers redistribution of nuclear p73 to the cytoplasm and, hence, suppresses its transcriptional activity. In addition, we show that cytoplasmic p73 contributes to the proapoptotic activity of Wwox. Our findings reveal a functional cross-talk between p73 and Wwox tumor suppressor protein.

Rearrangements of the ALL-1/MLL1 gene underlie the majority of infant acute leukaemias, as well as of therapy-related leukaemias developing in cancer patients treated with inhibitors of topoisomerase II, such as VP16 and doxorubicin. The rearrangements fuse ALL-1 to any of >50 partner genes or to itself. Here, we describe the unique features of ALL-1-associated leukaemias, and recent progress in understanding molecular mechanisms involved in the activity of the ALL-1 protein and of its Drosophila homologue TRITHORAX.

The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.

Since the majority of high-grade breast cancers express reduced levels of BRCA1 mRNA, we investigated the factors regulating BRCA1 transcription. Factors with specific affinity for the previously identified positive regulatory region (PRR) in the BRCA1 promoter were purified from whole-cell extracts. Identified proteins included replication protein A and a series of related factors with affinity for the sense strand of PRR. A subset of the identified factors activated the BRCA1 promoter. Identification of these families of proteins regulating the BRCA1 promoter represents an important step in the comprehension of the mechanisms responsible for breast cancer development.

ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of > or =29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter.

Overexpression of the TCL1 oncogene has been shown to play a causative role in T cell leukemias of humans and mice. The characterization of Tcl1-deficient mice in these studies indicates an important developmental role for Tcl1 in early embryogenesis. In wild-type embryos, Tcl1 is abundant in the first three mitotic cycles, during which it shuttles between nuclei and the embryo cortical regions in a cell-cycle-dependent fashion. The absence of this protein in early embryogenesis results in reduced fertility of female mice. The present studies elucidate the mechanism responsible for the reduced female fertility through analysis of the oogenesis stages and early embryo development in Tcl1-deficient mice. Even though Tcl1(-/-) females display normal oogenesis and rates of oocyte maturation/ovulation and fertilization, the lack of maternally derived Tcl1 impairs the embryo's ability to undergo normal cleavage and develop to the morula stage, especially under in vitro culture conditions. Beyond this crisis point, differentiative traits of zygotic genome activation and embryo compaction can take place normally. In contrast with this unanticipated role in early embryogenesis, we observed an overexpression of TCL1 in human seminomas. This finding suggests that TCL1 dysregulation could contribute to the development of this germinal cell cancer as well as lymphoid malignancies.

Members of the TCL1 family of oncogenes are abnormally expressed in mature T-cell leukemias and B-cell lymphomas. The proteins are involved in the coactivation of protein kinase B (Akt/PKB), a key intracellular kinase. The sequences and crystal structures of three Tcl1 proteins were analyzed in order to understand their interactions with Akt/PKB and the implications for lymphocyte malignancies. Tcl1 proteins are approximately 15 kD and share 25-80% amino acid sequence identity. The tertiary structures of mouse Tcl1, human Tcl1, and Mtcp1 are very similar. Analysis of the structures revealed conserved semi-planar surfaces that have characteristics of surfaces involved in protein-protein interactions. The Tcl1 proteins show differences in surface charge distribution and oligomeric state suggesting that they do not interact in the same way with Akt/PKB and other cellular protein(s).

The TCL1 gene at 14q32.1 is involved in chromosomal translocations and inversions in mature T cell leukemias. 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. In transgenic animals, the deregulated expression of TCL1 leads to mature T cell leukemia, demonstrating the role of TCL1 in the initiation of malignant transformation in T cell neoplasia. Expression of high levels of Tcl1 have also been found in a variety of human tumor-derived B cell lines ranging from pre-B cell to mature B cell. Here we describe the phenotype of transgenic mice, E mu-TCL1, established with TCL1 under the control of a V(H) promoter-Ig(H)-E mu enhancer to target TCL1 expression to immature and mature B cells. Flow cytometric analysis reveals a markedly expanded CD5(+) population in the peritoneal cavity of E mu-TCL1 mice starting at 2 mo of age that becomes evident in the spleen by 3-5 mo and in the bone marrow by 5-8 mo. Analysis of Ig gene rearrangements indicates monoclonality or oligoclonality in these populations, suggesting a preneoplastic expansion of CD5(+) B cell clones, with the elder mice eventually developing a chronic lymphocytic leukemia (CLL)-like disorder resembling human B-CLL. Our findings provide an animal model for CLL, the most common human leukemia, and demonstrate that deregulation of the Tcl1 pathway plays a crucial role in CLL pathogenesis.

FEZ1/LZTS1 is a tumor suppressor gene that maps to chromosome 8p22, a chromosomal region frequently deleted in many human malignancies, including transitional cell carcinoma (TCC) of the urinary bladder. FEZ1/LZTS1 alterations have been reported in esophageal, breast, prostate, and gastric carcinomas. Fez1 expression was studied in five TCC-derived cancer cell lines by Western blot analysis and in 60 primary TCCs of the urinary bladder by immunohistochemistry. Fez1 protein was absent or reduced in four of five cell lines and in 37 of 60 primary TCC examined. We also restored Fez1 protein expression in human SW780 TCC-derived cells lacking endogenous Fez1 protein to study the effects of Fez1 expression on cell proliferation, cell kinetics, and tumorigenicity in BALB/c nude mice. In vitro transduction of SW780 Fez1-negative cell, with Ad-FEZ1, inhibited cell growth, altered cell cycle progression, and suppressed subcutaneous tumor growth in nude mice. These results suggest that FEZ1/LZTS1 gene plays a role in the development of TCC of the urinary bladder by acting as a bona fide tumor suppressor gene both in vitro and in vivo.

Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators, which is required to maintain active states of Hox gene expression during development. We have purified from Drosophila embryos a trithorax acetylation complex (TAC1) that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression.

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 FEZ1/LZTS1 gene maps to chromosome 8p22, a region that is frequently deleted in human tumors. Alterations in FEZ1/LZTS1 expression have been observed in esophageal, breast, and prostate cancers. Here, we show that introduction of FEZ1/LZTS1 into Fez1/Lzts1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S-G(2)/M stage of the cell cycle. Fez1/Lzts1 protein is hyperphosphorylated by cAMP-dependent kinase during cell-cycle progression. We found that Fez1/Lzts1 is associated with microtubule components and interacts with p34(cdc2) at late S-G(2)/M stage in vivo. Present data show that FEZ1/LZTS1 inhibits cancer cell growth through regulation of mitosis, and that its alterations result in abnormal cell growth.

Purpose: Loss of heterozygosity (LOH) involving the short arm of chromosome 8 (8p) is a common feature of the malignant progression of human tumors, including gastric cancer. We have cloned and mapped a candidate tumor suppressor gene, FEZ1/LZTS1, to 8p22. Here we have analyzed whether FEZ1/LZTS1 alterations play a role in the development and progression of gastric carcinoma., Experimental Design: We examined Fez1/Lzts1 expression in 8 gastric carcinoma cell lines by Western blot, and in 88 primary gastric carcinomas by immunohistochemistry. Twenty-six of these 88 primary gastric carcinomas were also microdissected and tested for LOH at the FEZ1/LZTS1 locus and for mutation of the FEZ1/LZTS1 gene. Furthermore, we studied the FEZ1/LZTS1 gene regulation and transcriptional control and the methylation status of the 5' region of the gene in all 8 gastric carcinoma cell lines., Results: Fez1/Lzts1 protein was barely detectable in all of the gastric cancer cell lines tested and was absent or significantly reduced in 39 of the 88 (44.3%) gastric carcinomas analyzed by immunohistochemistry, with a significant correlation (P < 0.001) to diffuse histotype. DNA allelotyping analysis showed allelic loss in 3 of 17 (18%) and microsatellite instability in 4 of 17 (23.5%) cases informative for D8S261 at the FEZ1/LZTS1 locus. When we compared the presence of LOH with Fez1/Lzts1 expression, we found loss of protein expression in all three of the tumors with allelic imbalance at D8S261. A missense mutation was detected in one case that did not express Fez1/Lzts1. Hypermethylation of the CpG island flanking the Fez1/Lzts1 promoter was evident in six of the eight cell lines examined as well as in the normal control., Conclusions: Our findings support FEZ1/LZTS1 as a candidate tumor suppressor gene at 8p in a subtype of gastric cancer and suggest that its inactivation is attributable to several factors including genomic deletion and methylation.

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.

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an approximately 10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.

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 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.

We have identified a gene at 11q23, telomeric to MLL, that encodes a guanine nucleotide exchange factor (GEF). This gene is transcribed into a 9.5-kb mRNA containing a 4.6-kb ORF. By Northern analysis, it was found to be expressed in all human tissues examined including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon, and minimally in thymus. Analysis of the predicted protein sequence indicates that it has strong homology to several members of the family of Rho GEFs that includes such oncogenes as Dbl, Vav, Tiam, and Bcr. A patient with primary acute myeloid leukemia (AML) and a karyotype of 51,XY,+8,+19,+3mar was found to have the 5' end of MLL at exon 6 fused in-frame with the 3' end of almost the entire ORF of this gene, which we named LARG for leukemia-associated Rho GEF. Transcriptional orientation of both genes at 11q23 is from centromere to telomere, consistent with other data that suggest the MLL-LARG fusion resulted from an interstitial deletion rather than a balanced translocation. LARG does not appear to have any homology with other MLL partner genes reported thus far. Thus, LARG represents an additional member of the GEF family and a novel MLL fusion partner in acute myeloid leukemia.

Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias., (Copyright 2000 Wiley-Liss, Inc.)

The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion. Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations. However, the function of these motifs is still not clear. Studying ALL-1 presents an additional challenge because the gene is the human homologue of Drosophila trithorax. The latter is a member of the trithorax-Polycomb gene family which acts to determine the body pattern of Drosophila by maintaining expression or repression of the Antennapedia-bithorax homeotic gene complex. Here we apply yeast two hybrid methodology, in vivo immunoprecipitation and in vitro 'pull down' techniques to show self association of the SET motifs of ALL-1, TRITHORAX and ASH1 proteins (Drosophila ASH1 is encoded by a trithorax-group gene). Point mutations in evolutionary conserved residues of TRITHORAX SET, abolish the interaction. SET-SET interactions might act in integrating the activity of ALL-1 (TRX and ASH1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes.

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.

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.

We have cloned a gene, BDP, encoding a protein with homology to the retinoblastoma-binding proteins Rbp1 and Rbp2. It also has homology to DNA-binding proteins such as Bright, a B-cell-specific trans-activator, and the Drosophila melanogaster dead ringer gene product. Like MyoD, Bdp binds to the COOH-terminal region of pRb through its conserved region and to hypophosphorylated pRb. It also binds to the MAR of the immunoglobulin heavy-chain locus. Thus Bdp may contribute to the transcriptional regulation of genes involved in differentiation and tissue-specific expression.

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

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.

MutS homologues have been identified in nearly all organisms examined to date. They play essential roles in maintaining mitotic genetic fidelity and meiotic segregation fidelity. MutS homologues appear to function as a molecular switch that signals genomic manipulation events. Here we describe the identification of the human homologue of the Saccharomyces cerevisiae MSH5, which is known to participate in meiotic segregation fidelity and crossing-over. The human MSH5 (hMSH5) was localized to chromosome 6p22-21 and appears to play a role in meiosis because expression is induced during spermatogenesis between the late primary spermatocytes and the elongated spermatid phase. hMSH5 interacts specifically with hMSH4, confirming the generality of functional heterodimeric interactions in the eukaryotic MutS homologue, which also includes hMSH2-hMSH3 and hMSH2-hMSH6.

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

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.

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 ALL-1 gene is involved in human acute leukemia through chromosome translocations or internal rearrangements. ALL-1 is the human homologue of Drosophila trithorax. The latter is a member of the trithorax group (trx-G) genes which together with the Polycomb group (Pc-G) genes act as positive and negative regulators, respectively, to determine the body structure of Drosophila. We have cloned a novel human gene, ALR, which encodes a gigantic 5262 amino acid long protein containing a SET domain, five PHD fingers, potential zinc fingers, and a very long run of glutamines interrupted by hydrophobic residues, mostly leucine. The SET motif, PDH fingers, zinc fingers and two other regions are most similar to domains of ALL-1 and TRX. The first two motifs are also found in other trx-G and Pc-G proteins. The ALR gene was mapped to chromosome band 12q12-13, adjacent to the VDR gene. This region is involved in duplications and translocations associated with cancer. The analysis of ALR expression showed that its approximately 18 kb long mRNA is expressed, like ALL-1, in most adult tissues, including a variety of hematopoietic cells, with the exception of the liver. Whole mount in situ hybridization to early mouse embryos indicates expression in multiple tissues. Based on similarities in structure and expression pattern, ALR is likely to play a similar role to ALL-1 and trx, although its target genes have yet to be identified.