T-cell oncogene rhombotin-2 interacts with retinoblastoma-binding protein 2.

The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/Lmo2) has distinct functions in erythropoiesis and in T-cell leukemogenesis. Additional functions for RBTN2 are indicated by its expression in non-hematopoietic tissues. These diverse functions of RBTN2 are presumed to be accomplished through physical interaction with different protein partners that bind the LIM domains of RBTN2. To identify these proteins which may modulate the activity of RBTN2, a human cDNA library was screened using the yeast two-hybrid assay. Using the RBTN2 LIM domain region as 'bait', the retinoblastoma-binding protein 2 (RBP2) was identified as a partner for RBTN2. The interaction between RBTN2 and RBP2 was confirmed using in vitro binding assays, and by co-immunoprecipitation of the two proteins. Deletion analysis showed the second LIM domain of RBTN2 was necessary and sufficient for binding to the last 69 amino acids of RBP2. The interaction between RBTN2 and RBP2 had a functional consequence: the combination of RBP2 and RBTN2 gave higher transcription in vitro, than RBTN2 alone. The interaction with RBP2 suggests two additional functions for RBTN2: (i) RBTN2 may directly affect the activity of RBP2, and/or (ii) RBTN2 may indirectly modulate the functions of the retinoblastoma protein by binding to RBP2.

Characterization of immunoglobulin heavy chain genes from an acute lymphocytic leukemia with four rearrangements.

Approximately 25% of acute leukemias of the B-cell lineage demonstrate more than two rearranged immunoglobulin heavy chain genes when examined by Southern blot analysis. The origin of the extra bands was investigated by molecular cloning and sequencing of four rearranged genes from one patient's leukemic cells. All four rearrangements were apparently derived independently. Two of the rearrangements used the VH6 variable region, attached to different diversity and joining regions. One of the two rearrangements contained a mutation in the coding sequence leading to the generation of a nonsense codon. This rearranged gene also differed from the other VH6 containing gene starting at about 330 bp upstream of the ATG initiation codon. The third rearranged gene used a member of the VH2 variable gene family. A DH-JH rearrangement was found in the fourth rearranged gene. The data indicate that the leukemia probably arose as a result of the transformation of an early B-cell progenitor that lacked rearranged immunoglobulin genes but retained some differentiation potential.

Identification of genes potentially involved in LMO2-induced leukemogenesis.

The most common translocations in childhood T cell acute lymphoblastic leukemias involve the LMO2 locus on chromosome 11p13 and cause ectopic expression of the LMO2 gene in thymocytes. Transgenic mice with enforced expression of LMO2 in their thymocytes develop T cell leukemias thus demonstrating the role of LMO2 in leukemogenesis. The physiologic and leukemogenic functions of LMO2 are mediated through its transcriptional regulatory activities, but the identity of the target genes is completely unknown. In this report, we have used cDNA representational difference analysis (cDNA-RDA) to identify genes that are over-expressed and are likely to play a role in the LMO2 induced leukemias. cDNA-RDA was performed using very small amounts of mRNA pool (from 1 microg of total RNA) to reverse transcribe the cDNAs from leukemic cells or normal thymocytes. The cDNA-RDA led to the isolation of nine distinct clones that were specifically overexpressed in the leukemic cells. Sequence analysis revealed that five of the nine clones had identity or homology to known genes that are known to play a role in the pathogenesis of leukemias or other cancers. Three clones had no significant homology to any known genes and thus represent novel candidate genes. Our study demonstrates that cDNA-RDA using very small amounts of total RNA is a highly efficient method to identify novel genes that may play a role in leukemogenesis.

Disruption of T-cell differentiation precedes T-cell tumor formation in LMO-2 (rhombotin-2) transgenic mice.

Transgenic mice expressing LMO-2 (rhombotin-2) were constructed by placing the LMO-2 gene under control of the metallothionein promoter. Thymic tumors developed in approximately 15% of the transgenic mice between 37 and 71 weeks. Only T-cell tumors were found in the transgenic mice despite high expression of LMO-2 in all tissues. The thymic tumors were aggressive and were invariably associated with metastasis to non-lymphoid organs. In approximately 50% of apparently healthy transgenic mice there was up to a 10-fold expansion of CD4-CD8- double negative (DN) cells. Expansion of the DN cells was accompanied by the compensatory decrease in CD4+CD8+ double positive (DP) cells, indicating that breach of homeostasis within the thymus had not occurred in these animals. The increase in DN cells was associated with a clonal expansion of thymocytes, and increased proliferation within the thymus. Our data indicate that the ectopic expression of LMO-2 in T-cells disrupts normal T-cell differentiation by selectively expanding the DN thymocyte population prior to breach of homeostasis and overt leukemia/lymphoma.

Elf-2, a rhombotin-2 binding ets transcription factor: discovery and potential role in T cell leukemia.

Rhombotin-2 (RBTN-2) is a proto-oncogene only in the context of T lymphocytes. We postulated that the oncogenic effect of RBTN-2 in T cells is likely mediated by binding protein(s) with T cell-specific expression. By screening a T cell cDNA library, we identified a novel ets transcription factor that binds RBTN-2. This protein was named elf-2 because its DNA-binding domain is virtually identical to that of ets family member elf-1. Northern analyses showed similar levels of two elf-2 transcripts (3.5 kb and 3.8 kb) in all tissues except thymus. Thymocytes expressed four- to 10-fold greater amounts of the 3.5 kb transcript than other tissues. Sequence analyses of cDNA clones indicated that these transcripts encode proteins differing only at their amino termini, and likely represent alternatively spliced isoforms. These isoforms (elf-2a and elf-2b) contain identical RBTN-2 binding regions and DNA-binding domains. Elf-2b lacks a putative transactivation domain. The expression patterns suggest that RBTN-2 normally interacts equally with elf-2a and elf-2b. In contrast, when RBTN-2 is inappropriately expressed in T cells, RBTN-2 would interact predominantly with elf-2b; this interaction may lead to T cell proliferation.

Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome.

We investigated the level of minimal residual disease (MRD) in 26 children with B-lineage acute lymphoblastic leukemia (ALL) after intensive induction therapy. A quantitative semi-nested polymerase chain reaction (PCR) detecting the clone-specific rearranged immunoglobulin heavy chain genes was developed to improve sensitivity and specificity of amplification. In all patients, one leukemic cell could be detected in a background of 10(5) normal blood mononuclear cells. All patients investigated were in complete remission at the end of induction therapy as evaluated by morphologic criteria. Nineteen patients (73%) had no detectable residual leukemic cells using the sensitive semi-nested PCR. Seven patients (27%) were PCR positive. Three had a low level (<2 x 10(-5) leukemic cells per bone marrow cell), while four patients had a high level (>2 x 10(5)) of detectable residual leukemic cells. All patients with low or undetectable levels of residual leukemia remained in complete remission at a median of 63 months from diagnosis (range 40-80 months), while all four patients with a high level of residual leukemia subsequently relapsed at a median of 21 months from diagnosis (range 13-37 months). The patient groups with undetectable or low, and high level of MRD did not differ significantly in other clinical or genetic features with prognostic significance. We conclude that the level of MRD at the end of the intensive induction therapy period is predictive of outcome in childhood B lineage ALL. If confirmed by large prospective studies, the level of MRD might be useful in stratifying patients into high and low risk categories.

Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Minimal residual disease (MRD) is an independent prognostic factor in childhood acute lymphoblastic leukemia (ALL). The most widely applied MRD assays in ALL are flow cytometric identification of leukemia immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. We measured MRD by both assays in 227 patients with childhood B-lineage ALL. Of 1375 samples (736 bone marrow and 639 peripheral blood) examined, MRD was <0.01% in 1200, and > or =0.01% in 129 by both assays; MRD levels measured by the two methods correlated well. Of the remaining 46 samples, 28 had MRD > or =0.01% by flow cytometry but <0.01% by PCR. However, PCR (which had a consistent sensitivity of 0.001%) detected leukemic gene rearrangements in 26 of these 28 samples. Conversely, in 18 samples, MRD was > or =0.01% by PCR but <0.01% by flow cytometry. In nine of these samples, flow cytometry had a sensitivity of 0.001%, and detected aberrant immunophenotypes in eight samples. Therefore, the two most widely used methods for MRD detection in ALL yield concordant results in the vast majority of cases, although the estimated levels of MRD may vary in some. The use of the two methods in tandem ensures MRD monitoring in all patients.

Molecular evidence for minimal residual bone marrow disease in children with 'isolated' extra-medullary relapse of T-cell acute lymphoblastic leukemia.

Central nervous system (CNS) relapse confers a poor prognosis in children with acute lymphoblastic leukemia (ALL). It is uncertain whether morphologically undetectable leukemia is present in the bone marrow at the time of CNS relapse, or whether the CNS acts as a 'sanctuary' site to allow reseeding of the marrow at a later time. We examined DNA from bone marrow samples from six patients with T-cell ALL with isolated CNS relapse using sensitive polymerase chain reaction (PCR) assays to detect minimal residual disease. One of these PCR assays was based on amplification of leukemia-specific TCR-delta gene rearrangements, while the other assay relied upon detection of the c-tal deletion. In four patients, where bone marrow samples were taken at the time of CNS relapse, residual disease was detectable in every sample at a level below morphological detection. In addition, three patients had residual disease detected in their subsequent bone marrow when CNS disease was not evident. Our findings, although preliminary, suggest that relapse of leukemia in the CNS reflects resurgence of the disease in the bone marrow that is first detected clinically in the CNS. The concomitant molecular detection of bone marrow leukemia at time of 'isolated' CNS relapse in children with T-cell ALL explains subsequent bone marrow relapse in some of these children, and argues for intensive systemic therapy of these patients.

Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Children with acute lymphoblastic leukemia (ALL) with > or = 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10(4) or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels > or = 1 in 10(4). The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels < 1 in 10(4). In only two samples results were discordant: 2 in 10(4) and 5 in 10(4) leukemic cells by PCR but < 1 in 10(4) by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.

Quantification of minimal residual disease in T-lineage acute lymphoblastic leukemia with the TAL-1 deletion using a standardized real-time PCR assay.

Hematologic relapse remains the greatest obstacle to the cure of children with acute lymphoblastic leukemia (ALL). Recent studies have shown that patients with increased risk of relapse can be identified by measuring residual leukemic cells, called minimal residual disease (MRD), during clinical remission. Current PCR methods, however, for measuring MRD are cumbersome and time-consuming. To improve and simplify MRD assessment, we developed a real-time quantitative PCR (RQ-PCR) assay for detection of leukemic cells that harbor the TAL-1 deletion. We studied serial dilutions of leukemic DNA and found the assay had a sensitivity of detection of one leukemic cell among 100,000 normal cells. We then investigated 23 samples from eight children with ALL in clinical remission. We quantified residual leukemic cells by using the TAL-1 RQ-PCR assay and by using limiting dilution analysis. In 17 samples, both methods detected MRD levels > or =0.001%. The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.926). In the remaining six samples, both methods detected fewer than 0.001% leukemic cells. We conclude the TAL-1 RQ-PCR assay can be used for rapid, sensitive and accurate assessment of MRD in T-lineage ALL with the TAL-1 deletion.

Expression of the V(D)J recombinase gene RAG-1 is tightly regulated and involves both transcriptional and post-transcriptional controls.

The V(D)J recombinase activating genes, RAG-1 and RAG-2, are coexpressed only in immature lymphocytes, and are sufficient and necessary for V(D)J recombination to occur in non-lymphoid cells. In order to examine control mechanisms operative in the regulation of RAG-1 and RAG-2, we have studied the pattern of expression of these genes in human pre-T cells, pre-B cells, and thymocytes treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA); an agent which mimics some of the lymphocyte maturation changes seen in vivo. The expression of RAG-1 and RAG-2 was tightly controlled in a rapid, yet very complex, manner with both positive and negative control elements operating. Treatment of immature lymphocytes with TPA caused the specific and rapid elimination of steady-state RAG-1 and RAG-2 RNA. Nuclear run-on assays showed that TPA completely repressed the transcription of RAG-1 within 30 min. In addition to repressing the transcription of RAG-1, TPA treatment caused the rapid and specific degradation of RAG-1 transcripts by decreasing the apparent half-life of RAG-1 mRNA more than two-fold. As judged by cycloheximide treatment of cells, the effects of TPA were not dependent on new protein synthesis. A labile transcriptional repressor, separate from the TPA-associated repression of transcription, was also active in cells transcribing RAG-1 and RAG-2 RNA. After depletion of this labile repressor by cycloheximide treatment, steady-state RAG-1 and RAG-2 RNA levels, and their transcription rates, were elevated four- to six-fold; but were still susceptible to elimination by TPA treatment. Treatment of pre-T CEM cells with interleukin-2, or theophylline (an agent that increases intracellular cAMP) resulted in a two-fold increase in RAG-1 RNA suggesting that lymphokines, either independently or through second messengers, may modulate RAG-1 and RAG-2 expression. The complex, rapid and precise regulation of RAG-1 and RAG-2 expression is consistent with the view that it is necessary for the cell to tightly regulate V(D)J recombinase levels; lower expression may result in inefficient recombination of Ig/TCR genes, whereas increased expression may lead to recombination errors that are deleterious to the cell.

A human lymphoid leukemia cell line with a V(D)J recombinase-mediated deletion of hprt.

Large deletions of exons 2 and 3 of the hprt gene are the most common type of hprt mutation in lymphocytes of newborn infants, and their frequency increases in cultured human T-lymphoid cells as a result of exposure to etoposide. Sequenced PCR products for these deletions are consistent with a V(D)J recombinase-mediated mechanism underlying their genesis. Herein, we describe the isolation and characterization of an etoposide-induced mutant CEM cell line that is clonal for a V(D)J recombinase-mediated exon 2 + 3 deletion. Human CCRF-CEM cells were exposed to 5 muM etoposide for 4 h, selected in 6-thioguanine, and an exon 2 + 3 deletion mutant was isolated through serial limiting dilution, using a PCR-based assay for detection of the exon 2 + 3 deletion. Untreated CEM cells and cells treated with 6-thioguanine alone were similarly subcultured. The exon 2 + 3 deletion-containing line was termed SJCEM808 and had a slightly longer doubling time than the control lines, tended to clump in suspension, and was characterized by cell membrane blebbing. Compared to the parent line, SJCEM808 had similar cytogenetic abnormalities, lower CD2, CD1, and CD10 expression, and negligible RAG-1 expression. However, RAG-1 expression was down-regulated in some untreated parental subclones following similar subculturing. The sequence of the exon 2 + 3 deletion mutation exhibited nucleotide insertions, and the breakpoints were adjacent to heptamer signal recognition sequences in intact hprt, consistent with a V(D)J recombinase-mediated mechanism underlying its genesis. There were no MLL gene or interlocus T-cell receptor (TCR) rearrangements. These results indicate that non-homologous recombination following etoposide treatment is neither necessarily accompanied by other large DNA rearrangements nor simply a pre-lethal event, and this cell line may serve as a useful tool for studying illegitimate V(D)J recombinase-mediated deletions.

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA.

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.

mRNA transcripts initiating within the human immunoglobulin mu heavy chain enhancer region contain a non-translatable exon and are extremely heterogeneous at the 5' end.

Transcription events are thought to precede gene rearrangement in the immunoglobulin (Ig) loci and may be the mechanism by which the various gene regions are made accessible for recombination. If this is the case, identification and characterization of transcripts from the Ig loci should permit a better understanding of the gene rearrangement process. We have isolated a 2.3 kb cDNA clone from the human pre-B cell line Nalm-1 that contains enhancer-specific sequences from the Ig heavy (H) chain gene locus. The 2.3 kb transcript initiated within the enhancer region and showed extreme 5' heterogeneity, with more than 50 initiation sites mapping near the Ig-specific octamer ATTTGCGT. Sequencing of the cDNA clone demonstrated that 644 nucleotides from the Ig enhancer region were incorporated as a leader exon spliced to the mu constant (Cmu) region. This leader exon contained many translation termination codons and may function to inhibit the translation of sterile Cmu polypeptides. Using an enhancer-derived probe, we detected two low-abundancy mRNA transcripts with sizes of 2.3 and 12 kb. Northern blot analysis suggested that the 12 kb transcript was the unspliced precursor mRNA of a VDJ rearrangement. The potential role of these enhancer-containing transcripts in the opening of the IgH chain gene for rearrangement and for class switching is discussed.

Biochemical analysis of adenovirus type 5 DNA-binding protein mutants.

We previously reported the isolation and functional characterization of seven adenovirus type 5 (Ad5) DNA-binding protein (DBP) point mutants (Quinn, C. O., and Kitchingman, G. R. (1986) J. Virol. 60, 653-661). Six of the seven mutants were defective in their ability to help adeno-associated virus replicate its DNA. To determine the level at which the mutations affect this function of the DBP, we analyzed several properties of the mutant proteins. All are transported to the nucleus and are post-translationally phosphorylated to the same extent. The half-lives of the proteins, measured by pulse-chase, were nearly identical to that of the wild-type DBP. The mutant DBPs were examined for their ability to bind to single-stranded DNA (ssDNA). Mutations in amino acids 322, 323, and 470 lowered the affinity of the DBP for ssDNA, while a mutation in amino acid 181 had no affect. Combinations of mutations in amino acid 470 with either 322 or 323 did not further lower the affinity of the protein for ssDNA. These data indicate that the functional defect for adeno-associated virus helper activity of the six mutants is due mainly, if not totally, to their reduced affinity for single-stranded DNA. These experiments have thus identified a functional domain of the adenovirus type 5 DBP potentially involved in DNA-protein interactions. Comparisons with temperature-sensitive DBP mutants indicate that the conserved region mutants are functionally distinct and represent a new class of DBP mutants.

Uptake and utilization of deoxynucleoside 5'-monophosphates by Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual capacity for the uptake and utilization of exogenous deoxyribonucleoside 5'-monophosphates intact without prior dephosphorylation. In this study, it was found that once inside the cell, deoxyribonucleoside 5'-monophosphates were rapidly phosphorylated to the triphosphate level and incorporated into DNA. Catabolism of deoxyribonucleoside 5'-monophosphates was also observed. Competition studies indicated that a single uptake system with a higher affinity for deoxyribonucleotides mediates the uptake of nucleoside 5'-monophosphates.

T-cell proto-oncogene rhombotin-2 is a complex transcription regulator containing multiple activation and repression domains.

The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/LMO2) is involved in many processes, including leukemogenesis and erythropoiesis. It is thought that the principle role of RBTN-2 in these processes is to regulate transcription. To examine the potential for RBTN-2 to modulate transcription, we constructed RBTN-2/GAL4 DNA-binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct. From these studies we identified a transcription activation domain within the NH2 terminus of RBTN-2. This activation domain was further localized within a proline-rich 19-amino acid region. A second activation domain of 11 amino acids was also identified. This domain was located within the COOH terminus of RBTN-2, and functioned in mammalian cells but not in yeast. Furthermore, the two LIM domains of RBTN-2 were shown to function as transcription repression domains. Each individual LIM domain acted as an independent transcription repression domain on a heterologous activation domain. However, in context of full-length RBTN-2, the LIM domains selectively repressed the NH2-terminal activation domain, but had no effect on the COOH-terminal domain. Overall, these results demonstrate that the T-cell oncogene RBTN-2 is a complex transcription factor possessing multiple transcription regulatory modules, including two activation domains and two repression domains.

Formylation of methionyl-transfer ribonucleic acid in Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides grows readily but does not formylate methionyl-transfer ribonucleic acid in a defined medium without an added formyl donor. Formylation occurs when the medium is supplemented with N5,N10-methenyltetrahydrofolate or N10-formyltetrahydrofolate, but not with folate.

Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.

Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.