TEL Gene Research Articles

Retroviral Insertional Mutagenesis Identifies Genes that Collaborate with NUP98-HOXD13 during Leukemic Transformation

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

The MN1 oncoprotein activates transcription of the IGFBP5 promoter through a CACCC-rich consensus sequence

The IGF-binding protein (IGFBP) family consists of six proteins that are expressed and secreted in different tissues. The proteins are regulators of physiological processes throughout the body by modulating the activity of IGF-I and IGF-II. In this article, we describe the coordinated expression of IGFBP5 and MN1 in meningiomas. MN1 is a transcriptional co-activator and we show that MN1 stimulates the IGFBP5 promoter in Hep3B cells. A CACCC-containing sequence, located 140 bp upstream of the transcription start site of the promoter, is required for MN1 action. This sequence matches with the CACCCAC consensus sequence that was selected in an oligonucleotide selection assay performed for MN1. The CACCC element has also been shown to be important for induction of the IGFBP5 promoter by retinoic acid (RA) and progesterone (Pg). We were unable to confirm the effect of Pg on the promoter in Hep3B and U2-osteosarcoma cells regardless of the presence of MN1. On the other hand, we show that induction of the promoter by RA depends on co-expressed MN1 in Hep3B cells. MN1TEL, a leukemia-related fusion protein containing parts of the MN1 and TEL (ETV6) genes, is capable of stimulating the IGFBP5 promoter but is unable to cooperate with RA in Hep3B cells. This suggests that the effects of RA can be negatively affected in leukemias caused by MN1TEL.

Prospective Analysis of TEL and MLL Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia: A Children's Oncology Group Study.

In a retrospective study, we previously demonstrated that TEL gene rearrangements, representing the TEL-AML1 fusion created by the t(12;21), conferred a favorable prognosis among children with newly diagnosed acute lymphoblastic leukemia (ALL). In 1996, we undertook a prospective study of patients greater than one year of age with B-precursor ALL consecutively enrolled on the ALinC16 study to confirm this result in an independent cohort of patients with extended follow up, to determine the incidence of TEL and MLL gene rearrangements, and to determine if TEL gene status should be used for risk classification. Among 928 cases studied, TEL gene rearrangements were detected in 244 (26%), including 29% of standard-risk and 21% of high-risk cases by the National Cancer Institute/Rome criteria. TEL-rearranged cases had a median age of 4.4 years (91% < 10 years) and a median presenting leukocyte count of 12 × 109/L (81% < 50 × 109/L). Cases with germline TEL had similar features, with a median age of 5.0 years and a median presenting leukocyte count of 12 × 109/L. At a median follow-up of 7.8 years, the 5-year event-free survival (EFS) ± SE for patients with TEL rearrangements was 86% ± 2%, compared to 72% ± 2% for those with germline TEL (p<0.0001). TEL rearrangements were associated with a favorable outcome among standard risk patients (5-yr EFS 88% ± 3% vs. 78% ± 2%, p=0.001) as well as high risk patients (5-yr EFS 81% ± 5% vs. 62% ± 3%, p=0.003). TEL rearrangements were associated with a favorable outcome among patients with rapid early responses to therapy (5-yr EFS 87% ± 2% vs. 75% ± 2%, p=0.0002), but the association did not attain statistical significance among those with slow early responses (5-yr EFS 71% ± 12% vs. 56% ± 6%, p=0.16). Overall, patients with TEL rearrangements had an outcome similar to that of patients with trisomies 4 and 10 (5-yr EFS 86% ± 2% vs. 82% ± 3%, p=0.18). By contrast, MLL rearrangements were detected in only 19 cases (2%) and the 5-yr EFS for patients with this genetic abnormality was 58% ± 11%, compared to 76% ± 1% for those with germline MLL (p=0.02). MLL rearrangements were associated with a significantly worse outcome among standard-risk patients (5-yr EFS 55% ± 2% vs. 82% ± 2%, p=0.01), but not among high-risk patients (5-yr EFS 63% ± 2% vs. 66% ± 3%, p=0.57). However, these findings should be interpreted with caution because of the small number of patients with MLL rearrangements in each risk group. The finding of TEL rearrangements in association with a favorable prognosis among both standard-risk and high-risk patients indicates that TEL gene status should be incorporated into current risk classification schemes for childhood ALL. We suggest that patients with TEL rearrangements and good early responses to therapy should be assigned to lower-risk treatment protocols.

Distinct Cytogenetic Aberrations during the Course of Mantle Cell Lymphoma (MCL).

BACKGROUND: Apart from t(11;14)(q13;q32), MCL is also characterized by other nonrandom cytogenetic findings. These additional aberrations are well studied at diagnosis and believed to represent clonal evolution during lymphomagenesis, but little is known about karyotypic changes during the course of the disease.METHODS: The study included 33 patients with MCL. In all cases, an interphase FISH assay was performed at diagnosis on lymphoma cells from peripheral blood, bone marrow or touch imprints of involved sites. Commercial probes were employed for the detection of t(11;14), +12, 13q−, and abnormalities of ATM, p53, p16, TEL, c-MYC and BCL6 genes. In 14 cases, the same FISH screening was repeated at least once (up to four times) during the course of the disease, at relapse or in the context of partial or no response.RESULTS: The most frequent additional findings at diagnosis were ATM deletion in 15 cases (45.5%) and 13q− in 12 cases (36.4%), followed by p16 deletion (3 cases; 1 homozygous), p53 deletion (2 cases), and +12, duplication of the CCND1/IGH fusion gene and BCL6 triplication, in one case each. 11 of the 14 cases studied at follow-up showed karyotypic evolution, with acquisition of p16 deletion (6 cases; 4 homozygous), TEL deletion (5 cases; 2 on the basis of monosomy 12), duplication of the CCND1/IGH fusion (3 cases), p53 deletion (2 cases), and c-MYC amplification (1 case). There was no case with acquisition of ATM deletion, 13q− or +12, but in two cases with 13q− in a minor subclone at diagnosis the aberration was estimated to involve the total of the lymphoma cells at relapse. Interestingly, new BCL6 aberrations were seen in 3 cases (triplication in one and amplification in the other two, including the case with gene triplication at diagnosis) and were detected at the third or the fourth repetition of the screening. The longest survival after detection of these aberrations was 3 months.CONCLUSIONS: Our data suggest that in most cases of MCL clonal evolution also occurs during the course of the disease, with the acquisition of multiple additional chromosomal lesions. Despite the small number of patients in our series, it seems that some of the aberrations (like ATM deletion or 13q−) are most commonly already present at diagnosis, while others (such as monosomy 12 and/or TEL deletion) appear more often or even exclusively on follow-up. From the clinical point of view, we found that the most informative finding is the overrepresentation of the BCL6 gene, apparently associated with aggressive behavior and perhaps the terminal stage of MCL.

TEL/AML1 Expression in a Non-B Cell Line Induces a Set of Differentially Expressed Genes, but Points to Cell Type Specific Effects of the Fusion Protein in Leukemic Blasts of TEL/AML1 Positive Patients.

Acute lymphoblastic leukemia (ALL) is the predominant malignancy in childhood (30%). The majority of ALL (75%) exhibit characteristic chromosomal aberrations with prognostic significance that are used to classify the heterogenic disease into subgroups. Among these, the translocation t(12;21) resulting in the fusion of the TEL and AML1 genes is the most frequent chromosomal rearrangement in childhood ALL (20 – 25%). Both genes code for transcription factors essential for normal hematopoiesis. Whereas AML1 functions mostly as an activator of expression, TEL/AML1 acts as a repressor in a dominant manner. To date, detailed knowledge on the effect of TEL/AML1 in the process of malignant transformation is widely lacking. To address the consequence of TEL/AML1 expression, we established stable TEL/AML1 expressing cell lines of B lymphoblastoid and non-B cell origin using an episomal vector system and performed microarray-based gene expression profiling. To this end, we obtained gene expression profiles of a TEL/AML1 expressing non-B cell clone and the corresponding empty vector control. We first set out to gain a measure for the comparability of gene expression profiles in leukemic cells of TEL/AML1 positive ALL patients and the TEL/AML1 expressing non-B cell line. We therefore used published gene expression data of initial and relapsed ALL to calculate diagnostic gene expression signatures that faithfully predict genetic and immunological ALL subtypes in patient samples. We then used these signatures to classify a test set of patient samples and the TEL/AML1 expressing non-B cell into these subtypes. All patient samples were predicted correctly, however, the TEL/AML1 non-B cell line was not classified as TEL/AML1 positive, indicating that the diagnostic signature of TEL/AML1 positive leukemic cells is not induced by TEL/AML1 in the non-B cell line. Nevertheless, we identified several genes differentially expressed in the non-B cell line as compared to the empty vector control. The majority of genes are down-regulated, in agreement with the repressor function of the fusion protein. Among these genes are several transcription factors and genes playing a role in hematopoietic development and immune response. Furthermore, we found several genes with a role in neuronal development and disease. Interestingly, TEL knock out mice die during embryonal development due to apoptosis of mesenchymal and neuronal cells, supporting a function of TEL in the nervous system. Thus, a set of genes is regulated by TEL/AML1 in a non-B cell line, compatible with the proposed functions of TEL and AML1. However, the diagnostic signature of leukemic blasts of TEL/AML1 positive ALL patients was not induced in these cells supporting the view that besides common changes in gene expression cell type specific effects of TEL/AML1 exist and contribute to leukemogenesis.

A t(12;17)(p13;q12) identifies a distinct TEL rearrangement-negative subtype of precursor-B acute lymphoblastic leukemia

Structural rearrangements involving the short arm of chromosome 12 are common in acute lymphoblastic leukemia (ALL) and often involve the TEL locus at 12p13. The balanced t(12;17)(p13;q12) is a rare but recurrent aberration in ALL. Whereas the TEL gene has been postulated as a likely candidate for involvement in the t(12;17), the precise molecular consequences of this translocation have not yet been elucidated. We identified a t(12;17) in 2 of 398 childhood ALL patients karyotyped at presentation in our institute. Both cases had a precursor-B immunophenotype and were CD10 negative and CD33 positive. Fluorescence in situ hybridization excluded involvement of the TEL locus in the t(12;17) and provided no evidence for concomitant cryptic deletion of the 12p commonly deleted region. Comparison of these and previously published cases demonstrates that the translocation predominately occurs in children and young adults with precursor B-ALL and is typically characterized by low CD10 expression and high CD33 expression. Our data support the involvement of a new locus telomeric to TEL in the pathogenesis of t(12;17)-positive ALL.

Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)-positive pediatric ALL, and their relation with drug sensitivity and clinical outcome

Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS +/- s.e. 53 +/- 17%) and of those with an extra der(21)t(12;21) (60 +/- 22%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (79 +/- 6%; P-trend = 0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to prednisolone (hazard ratio 5.78, 95% CI 1.45-23.0; P=0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL.

Deletion of Tumor Suppressor Genes (TSGS) in Patients with Myelodysplastic Syndrome (MDS).

Background: The deletion of genomic sites harboring TSGs is known to be a powerful prognostic indicator in various chronic hematologic malignancies. However, the clinical significance of TSGs loss in MDS has not been thoroughly investigated.Aims: To summarize our experience on the incidence and essential clinical correlates of TSGs deletions in MDS, providing preliminary results from the study of 27 patients.Methods: The study included 17 men and 10 women (median age 74; range 38–84 years) with a documented diagnosis of MDS. The distribution of FAB subtypes was RA in 13, RARS in 3, RAEB in 6, RAEB-T in 4 and CMML in 1 case. According to the IPSS stratification, 5 patients were characterized as “low-risk”, 16 as “intermediate-risk” (11 as INT-1 and 5 as INT-2) and 6 as “high-risk” patients. The diagnostic bone marrow smears were studied with fluorescence in-situ hybridization (FISH) for deletions of chromosome regions 9p21 (p16/p14 and p15 genes), 9q34, 12p13 (TEL gene), 13q14 (RB1 gene D13S319 locus) and 17p13 (p53 gene).Results: A deletion in at least one of the chromosome regions or loci studied was detected in 10 patients (37%). In 6 of them, more than one region locus was lost, making up a total of 20 deletions. The commonest finding was loss of the 17p13 region (5 cases; two of them homozygous), followed by 9p21 loss (5 cases; one of them homozygous). Overall, the presence of TSGs deletions was associated with complex karyotype, high IPSS score and risk of death (regardless of leukemic progression). Interestingly, deletion in at least one of the TSGs studied was found in 5 of the 17 patients presenting with normal karyotype. In 3 of these patients, the MDS progressed to acute leukemia. Two of the 5 patients died at one and six months from diagnosis of the MDS, without leukemic conversion.Summary/Conclusions: Deletions of TSGs are not uncommon in MDS. Interestingly, we have found that certain TSGs, such as the p16/p14 and p15 genes at 9p21, the loss of which is mostly involved in the initiation or progression of lymploid neoplasms, are also frequently deleted in MDS. Overall, TSGs deletion in this small group of patients is apparently associated with other adverse biological and clinical features and poor outcome. Therefore, these preliminary observations justify the expansion of the study in a larger patient cohort, in order to clarify if the loss of certain TSGs is an independent prognostic factor in MDS and, thus, may be of help in the initial diagnostic approach and risk stratification of the patients.

Secondary Cytogenetic Abnormalities and Outcome in Children with TEL-AML1-Positive Acute Lymphoblastic Leukemia.

The t(12;21) translocation, which results in the fusion of the TEL (ETV6) and AML1 (RUNX1) genes, is present in ~25% of pediatric B-precusor acute lymphoblastic leukemia (ALL) patients. Although initially thought to be a favorable prognostic indicator, the t(12;21) was associated with a similar rate of relapse as t(12;21)-negative pre-B ALL in subsequent studies. While secondary cytogenetic abnormalities are often present, their prognostic significance is unknown. The objective of this study is therefore to examine the type, frequency and prognostic impact of secondary cytogenetic abnormalites in t(12;21)-positive blasts of children with ALL. We studied retrospectively 56 patients diagnosed with t(12;21)-positive ALL at the Hospital for Sick Children between 2000–2005. The mean age at diagnosis was 4.9 years (range <1 to 12 years). Nine patients (16%) presented with a white blood cell count of greater than 50 × 109/L. Cytogenetic studies consisted of a combination of FISH, G-banding and spectral karyotyping. The most frequent secondary cytogenetic changes in patients with t(12;21)-positive ALL were: the deletion of the non-translocated TEL allele (38%), an extra copy of the TEL-AML1 fusion gene (14%). Additional numerical abnormalities were seen in approximately one-third of the cases, with the most common being a gain of chromosome 21(13%). Recurrent deletions of chromosomal regions 6q and 11q were observed (14%). A surprising degree of karyotypic complexity was noted. Three or more additional chromosome abnormalities, two abnormal clonal lines, and complex structural rearrangements involving TEL-AML1 were detected in 28%, 21%, and 9% of patients, respectively. Five children (9%) in this cohort have developed a relapse of ALL. The 5-year event-free survival (EFS) was 83±7%. The 5-year EFS for patients with a deleted second TEL allele or an extra TEL-AML1 fusion was not statistically different. Of note, 4 of the children who relapsed had high-risk features at presentation (NCI criteria). All were treated with chemotherapy protocols developed for lower risk ALL. The realization that TEL-AML1 does not always predict excellent prognosis indicates that an on-going study of patients and their survival, with detailed analysis of complex genetic changes, is necessary for a reliable assessment of the prognostic value of the t(12;21) in pediatric ALL.

The presence of TEL/AML1 rearrangement and cryptic deletion of the TEL gene in adult acute lymphoblastic leukemia (ALL)

TEL/AML1 (also known as ETV6/RUNX1) rearrangement is the most frequent genetic change in childhood B-acute lymphoblastic leukemia (ALL) and is associated with a favorable prognosis. Its presence in adult ALL, however, has been questionable, and any association between TEL/AML1 rearrangement and clinical prognosis is unknown. To reveal the presence and incidence of the TEL/AML1 rearrangement in adult ALL, we applied fluorescence in situ hybridization (FISH). We conducted extra-signal, dual-color fluorescence in situ hybridization (ES-FISH) for TEL/AML1 rearrangement on bone marrow cells from 74 adult ALL patients and analyzed the survival time. We demonstrated the TEL/AML1 rearrangement in 2 patients (2.7%) among 74 patients with ALL. Of 74 adult ALL patients, 3 (4.0%) showed deletion of the TEL gene without TEL/AML1 rearrangement. The mean survival time of patients with TEL/AML1 +ALL and patients with cryptic TEL deletion was 33 and 5 months, respectively, whereas the mean survival time of 53 TEL/AML1 −ALL patients was 16.7 months. TEL/AML1 rearrangement is not unique in childhood ALL, and cryptic TEL deletion without TEL/AML1 rearrangement was more frequent than the TEL/AML1 rearrangement in adult ALL. We recommend TEL/AML1 FISH in adult ALL patients because conventional cytogenetic studies so far have yielded uninformative results.

Cytogenetic Findings in Pediatric T-Lymphoblastic Lymphomas: One Institution's Experience and a Review of the Literature

Cytogenetic analyses of lymphomas commonly reveal nonrandom chromosomal abnormalities, but there are relatively few reports in childhood lymphoblastic lymphoma (LL). We retrospectively reviewed G-banded karyotypic analyses performed at Arkansas Children's Hospital between 1990 and 2004. Six children (2 to 20 years old) had LL that presented as mediastinal or cervical masses and had a T-cell immunophenotype and clonal abnormalities. The cytogenetic findings in these 6 patients were as follows: 46,XX,-7,inv(9)(p11q12),der (12)t(7;12)(q11.2;p13),t(16;18)(p13.1;q21),+22 in patient 1; 47,XX,+9,del(9)(q11q22)x2 in patient 2; 72-119, XY,+X,+1,+1, inv(2) (p11q13),-3,+5,+6,+7,+10,-12,-16, -21,-21,-22,+mar in patient 3; 48,XY,+5,+20,t(7;9) (q32;q34) in patient 4; 47 approximately 48,XX,der(10)t(10;14)(q23; q11.2),+12, del(12)(p12)x2, -14,del(16)(q22q22),+?add (19)(p13.3) in patient 5; and 48 approximately 49,XY,+7,+8,t(11;19) (q23;p?13.3),+der(19)t(11;19)[cp20] in patient 6. Eleven chromosome breakpoints in 6 of our patients (7q11, 12p13, 16p13, 18q21, 9q11, 2p11, 2q13, 7q32, and 7q23) have been reported in other patients with acute lymphoblastic leukemia or LL and involved regions containing TEL, ABL, E2A, MLL, and T-cell receptor-alpha genes. A review of the cytogenetic findings of these and other cases of LL reveals that clonal aberrations are common and most frequently involve T-cell receptor gene regions. The aberrations show some features similar to those of acute lymphoblastic leukemia and are not unique to LL, thus furnishing additional evidence of the equivalence of these two diseases. The cytogenetic features of LL may be helpful in the diagnosis of pediatric lymphomas and undifferentiated neoplasms.

Isolated del(14)(q21) in a case of precursor B-cell acute lymphoblastic leukemia

We present a case of del(14)(q21) as a sole abnormality in a 4-year-old boy diagnosed with precursor B-cell acute lymphoblastic leukemia (pre-B ALL). To our knowledge, this is the first case of isolated del(14)(q21) in pre-B ALL. Two pretreatment bone marrow samples obtained 5 days apart were analyzed by cytogenetics. The G-banded karyotypes of the two samples were similar, differing only in the ratio of normal/abnormal metaphases detected. Both samples showed a del(14)(q21) as the only abnormality. Fluorescence in situ hybridization performed using the probes TEL/AML1 and immunoglobulin heavy chain (IGH) showed no fusion involving the TEL and AML1 genes and only a single IGH signal in 20% of the interphase cells analyzed.

Cloning and Characterization of the Novel Chimeric Gene TEL/PTPRR in Acute Myelogenous Leukemia with inv(12)(p13q13)

We have cloned a novel TEL/protein tyrosine phosphatase receptor-type R (PTPRR) chimeric gene generated by inv(12)(p13q13). PTPRR is the first protein tyrosine phosphatase identified as a fusion partner of TEL. The chimeric gene fused exon 4 of the TEL gene with exon 7 of the PTPRR gene, and produced 10 isoforms through alternative splicing. Two isoforms that were expressed at the highest level in the leukemic cells could have been translated into COOH-terminally truncated TEL protein possessing the helix-loop-helix domain (tTEL) and TEL/PTPRR chimeric protein linking the helix-loop-helix domain of TEL to the catalytic domain of PTPRR. These two mutant proteins exerted a dominant-negative effect over transcriptional repression mediated by wild-type TEL, although they themselves did not show any transcriptional activity. Heterodimerization with wild-type TEL might be an underlying mechanism in this effect. TEL/PTPRR did not exhibit any tyrosine phosphatase activity. Importantly, overexpression of TEL/PTPRR in granulocyte macrophage colony-stimulating factor-dependent UT7/GM cells resulted in their factor-independent proliferation, whereas overexpression of tTEL did not. After cytokine depletion, phosphorylated signal transducers and activators of transcription 3 (STAT3) significantly declined in mock cells, but remained in both tTEL- and TEL/PTPRR-overexpressing cells. Loss of tumor suppressive function of wild-type TEL and maintenance of STAT3-mediated signal could at least partly contribute to the leukemogenesis caused by inv(12)(p13q13).

Expression patterns of TEL genes in Poaceae suggest a conserved association with cell differentiation

Poaceae species present a conserved distichous phyllotaxy (leaf position along the stem) and share common properties with respect to leaf initiation. The goal of this work was to determine if these common traits imply common genes. Therefore, homologues of the maize TERMINAL EAR1 gene in Poaceae were studied. This gene encodes an RNA-binding motif (RRM) protein, that is suggested to regulate leaf initiation. Using degenerate primers, one unique tel (terminal ear1-like) gene from seven Poaceae members, covering almost all the phylogenetic tree of the family, was identified by PCR. These genes present a very high degree of similarity, a much conserved exon-intron structure, and the three RRMs and TEL characteristic motifs. The evolution of tel sequences in Poaceae strongly correlates with the known phylogenetic tree of this family. RT-PCR gene expression analyses show conserved tel expression in the shoot apex in all species, suggesting functional orthology between these genes. In addition, in situ hybridization experiments with specific antisense probes show tel transcript accumulation in all differentiating cells of the leaf, from the recruitment of leaf founder cells to leaf margins cells. Tel expression is not restricted to initiating leaves as it is also found in pro-vascular tissues, root meristems, and immature inflorescences. Therefore, these results suggest that TEL is not only associated with leaf initiation but more generally with cell differentiation in Poaceae.

A Contribution to the Cytogenetic Study of Plasmacytoid Dendritic Cell Acute Leukemia.

BACKGROUND: Plasmacytoid dendritic cell (DC2) acute leukemia is a recently described hematological malignancy with distinct clinical and phenotypic features. Yet, with fewer than 40 cases studied so far, the cytogenetic profile of this entity is still elusive. Previous studies (Leroux et al, 2002) have revealed recurrent chromosomal aberrations affecting certain genomic targets and the analysis of additional cases is expected to clarify whether these aberrations - alone or in combination - may constitute specific markers for the disease.METHODS: Seven patients with a well-established diagnosis of DC2AL (based on clinical, morphological and phenotypic features) were included in the study. A conventional cytogenetic analysis (CCA) was performed on unstimulated bone marrow cultures. Additionally, the diagnostic marrow smears were studied with interphase fluorescent in-situ hybridization (FISH), using commercially available probes for 5q31, 6q21, 7q31, 9p21 (p16 gene), 9q34, 11q23 (MLL gene), 12p13 (TEL gene), 13q14, 17p13 (p53 gene) and 21q22 (AML1 gene) regions. In case of any abnormal finding, further study was performed with the use of centromeric or other appropriate arm/chromosome enumeration probe to distinguish between region deletion or overrepresentation and numerical aberrations of the carrier arm/chromosome.RESULTS: An abnormal karyotype (by either CCA or FISH) was documented in all seven cases studied, with most lesions indicating the loss of genomic material. The most common finding was a rearrangement of the 21q22 region (involving the AML1 gene). Deletions of 6q21, 9q34, 11q23, 12p13 and 13q14, trisomy 12 and trisomy of 13q were detected in only one case each. Interestingly, some aberrations, including the three cases of AML1 rearrangement, were missed on CCA, suggesting a submicroscopic abnormality.CONCLUSIONS: The study failed to define recurrent chromosomal lesions in DC2AL, other than 21q22 rearrangements. Our results point to a possible pathogenetic role of the AML1 gene. Since AML1 is involved in both myeloid and lymphoid malignancies, it makes a reasonable candidate for leukemogenesis also in the case of DC2AL. Additional investigation at the cytogenetic and molecular level is needed to verify this assumption.

Cloning and Characterization of a Novel Leukemogenetic Gene TEL-PTPRR.

The TEL gene mapped on 12p13 is rearranged by chromosomal translocations in a variety of human leukemias and is fused to various partner genes mainly encoding receptor type and non-receptor type tyrosine kinases and transcription factors. Using 3′ RACE method, we have cloned novel chimeric cDNAs TEL/protein tyrosine phosphatase receptor type R (PTPRR) that is generated by inv(12)(p13q13) found in an acute myeloid leukemia case. TEL is a member of ETS family transcription factors and contains the homodimerizing or heterodimerizing helix-loop-helix domain at the N-terminus and the DNA-binding ETS domain at the C-terminus. TEL works as a tumor suppressor. On the other hand, PTPRR is a brain-specific receptor type protein tyrosine phosphatase with a single intracellular catalytic domain. PTPRR is the first tyrosine phosphatase that was identified as a fusion partner for TEL. The chromosomal rearrangement joined exon 4 in the TEL gene and exon 7 in the PTPRR gene and produced ten isoforms of TEL/PTPRR cDNAs through alternative splicing. Among them, only one expresses TEL/PTPRR chimeric protein that consists of the HLH domain from TEL and the catalytic domain from PTPRR and the others express truncated forms of TEL. Because reciprocal PTPRR-TEL mRNA as well as wild-type-PTPRR mRNA was not detected by RT-PCR analysis, TEL-PTPRR is likely to be involved in leukemogenesis. To clarify molecular mechanisms by inv(12), we first molecularly analyzed the TEL/PTPRR chimeric cDNA and the N-terminally truncated TEL cDNA containing exons 1–4 (tTEL). When transiently expressed in NIH3T3 cells, both TEL/PTPRR and tTEL were equally contributed to the cytoplasm and the nucleus. Moreover, both isoforms were found to associate with wild-type-TEL by immunoprecipitation assays. Therefore, we speculated that TEL/PTPRR and tTEL affect wild-type-TEL's functions in the nucleus through heterodimerizing with it. As expected from their structures, both isoforms did not bind to the ETS binding consensus sequence (EBS). In reporter assays with (ETS)3tkLuc, they did not repress transcription through EBS, while wild-type-TEL did. Interestingly, both TEL/PTPRR and tTEL dominantly interfered with the wild-type-TEL-induced transcriptional repression in a dose-dependent manner. On in vitro phosphatase assays using p-NPP as a substrate, both isoforms were shown to completely lack phosphatase activities. To assess the biological properties of TEL/PTPRR, we employed human megakaryoblastic leukemia cell line UT7/GM that proliferate completely depending on granulocyte macrophage-colony stimulating factor (GM-CSF). Overexpression of TEL/PTPRR resulted in factor-independent proliferation in UT7/GM cells. Interestingly, phosphorylated STAT3 remained after GM-CSF withdrawal in the TEL/PTPRR-overexpressing cells, while it rapidly declined in the mock cells. All these data collectively suggest that appearance of inv(12) causes leukemic transformation in human myeloid cells, at least partly through inactivating tumor-suppressive functions of wild-type-TEL. Considering that STAT3-mediated signals are maintained even after the cytokine depletion when TEL/PTPRR is overexpressed, TEL/PTPRR may regulate JAK/STAT signals through unknown mechanisms.

The TEL/ARG leukemia oncogene promotes viability and hyperresponsiveness to hematopoietic growth factors.

The TEL/ARG oncogene associated with acute myeloid leukemia is formed by the t(1;12)(q25;p13) reciprocal translocation, which fuses part of the TEL gene to the tyrosine kinase, c-ARG. In an effort to determine the biological effects and investigate signaling of the TEL/ARG fusion protein, multiple sublines of Ba/F3 cells were generated in which a TEL/ARG complementary DNA was expressed under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, rapidly induced expression of the TEL/ARG protein. TEL/ARG was heavily phosphorylated on tyrosine residues and was also found to rapidly induce tyrosine phosphorylation of multiple cellular proteins, including rasGAP, CBL, STAT5, PI3K, SHP2, Dok, and SHC. The Ba/F3-tet-TEL/ARG cells remained interleukin (IL)-3 dependent without doxycycline but with doxycycline displayed a marked reduction in cell death in the absence of IL-3. TEL/ ARG cells also displayed an enhanced proliferative response to IL-3 and to insulin-like growth factor 1. At least in Ba/F3 cells, although the growth rate was much lower compared to that with IL-3, TEL/ARG appeared to induce some cell proliferation as an immediate consequence. Nonetheless, the hyperresponsiveness to growth factors reported here is more likely to contribute to the pathogenesis of leukemia.

TEL-Syk fusion constitutively activates PI3-K/Akt, MAPK and JAK2-independent STAT5 signal pathways

We previously reported the fusion of the TEL gene to the Syk gene in myelodysplastic syndrome with t(9;12)(q22;p12). TEL-Syk fusion transformed interleukin-3 (IL-3)-dependent murine hematopoietic cell line BaF3 to growth factor independence. Here, we investigate the intracellular signal transduction of the stable transfectants. TEL-Syk fusion protein was associated with the p85 subunit of phosphatidyl inositol 3 kinase (PI3-K) followed by the activation of Akt in the absence of IL-3. Vav, phospholipase C-gamma2 and mitogen-activated protein kinase (MAPK) were also constitutively activated. TEL-Syk also activated the signal transducer and activator of transcription 5 (STAT5) in the absence of Janus kinase 2 activation. None of these kinases were phosphorylated in the BaF3 cells transfected with TELDeltaPNT-Syk in which the oligomerization domain of TEL was deleted. Inhibitor analysis showed that the MAPK pathway was important in TEL-Syk-mediated cell proliferation. The immunofluorescence technique revealed that the TEL-Syk fusion protein was located in the cytoplasm. These data suggest that TEL-Syk fusion protein in the cytoplasm leads to the constitutive activation of PI3-K/Akt, MAPK and STAT5 signal pathways, which are closely involved in IL-3-independent cell proliferation of BaF3 cells.

Allelotype analysis in relapsed childhood acute lymphoblastic leukemia.

We performed for the first time the allelotype of relapsed childhood acute lymphoblastic leukemia (ALL). A total of 38 cases were screened for loss of heterozygosity (LOH) using 71 markers. In all, 26 (68%) patients showed LOH on at least one chromosomal arm, indicating that LOH is a frequent event at relapse. The most frequent loss was found on chromosomal arm 9p at the p16/INK4a locus (39%). LOH at the TEL gene locus on chromosomal arm 12p also occurred often (25%). Frequent loss was observed on chromosome arms 4q (20%), 6q (21%), and 17q (20%). Sequential analysis (i.e. samples obtained from both initial diagnosis and relapse) shows that some patients (63%) have the identical LOH status at both phases, suggesting the presence of the same clone. Other samples (37%) showed distinct LOH alterations, indicating clonal evolution at relapse. Despite the heterogeneous and complex changes, some shared LOH loci occurred in these matched samples, suggesting that many of the same tumor-suppressor genes are aberrant at both phases. In summary, novel tumor-suppressor genes on chromosome arms 4q, 6q, and 17q, as well as the p16 and TEL genes, have an important role in the relapse of childhood ALL.

A fluorescence in situ hybridization study of TEL- AML1 fusion gene in B-cell acute lymphoblastic leukemia (1984–2001)

Acute lymphoblastic leukemia (ALL) is associated with recurrent chromosomal abnormalities. The cryptic translocation t(12;21)(p13;q22), which leads to the TEL- AML1 fusion gene, is the most common abnormality in childhood B-cell ALL. The aim of this retrospective study was to determine the incidence of TEL- AML1 fusion in childhood and adult B-cell ALL using interphase fluorescence in situ hybridization (I-FISH) and its association with additional changes. FISH, using dual-color extra-signal (ES) DNA probe specific for the TEL and AML1 genes, was performed either on blast cells suspensions stored in liquid nitrogen immediately after Ficoll or on leukemic cells preserved in fixative solution at −20°C after short-term culture. No TEL- AML1 fusion was observed in the 26 ALL adults. The fusion was found among 19.6% of the 57 ALL children, additional changes being identified by conventional cytogenetics in 80% of the cases. A deletion of the untranslocated TEL was observed in 36.3% of the ALL with the TEL- AML1 fusion. The coexpression of myelocytic and B-lymphoid antigens was found in 3 of the 11 of TEL- AML1 fusion positive-ALL. Our results (frequency of TEL- AML1 fusion in children and of the deletion of the untranslocated TEL allele, mean age of the patients and white blood cell count) are within the range observed by others. Structural chromosomal abnormalities other than the t(12;21) are frequent and may play a role in the prognosis of these patients.