scid Thymocytes with TCRbeta gene rearrangements are targets for the oncogenic effect of SCL and LMO1 transgenes.

SCL and LMO1 were both discovered by virtue of their activation by chromosomaltranslocation in patients with T-cell acute lymphoblastic leukemia (T-ALL). Overexpression of SCL and LMO1 in the thymus of transgenic mice leads to T-ALL at a young age. scid (severe combined immunodeficient) mice are unable to efficiently recombine antigen receptor genes and consequently display a developmental block at the CD4-CD8- to CD4+CD8+ transition. To test the hypothesis that this developmental block would protect SCL/LMO1 transgenic mice from developing T-ALL, we crossed the SCL and LMO1 transgenes onto a scid background. The age of onset for T-ALL in the SCL/LMO1/scid mice was significantly delayed (P < 0.001) compared with SCL/LMO1/wild-type mice. Intriguingly, all of the SCL/LMO1/scid malignancies displayed clonal, in-frame TCRbeta gene rearrangements. Taken together, these findings suggest that the "leaky" scid thymocyte that undergoes a productive TCRbeta gene rearrangement is susceptible to the oncogenic action of SCL and LMO1 and additionally suggests that TCRbeta gene rearrangements may be required for the oncogenic action of SCL and LMO1.

Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.

We have established a panel of nine immortal cell lines from T cell malignancies which arose in mice transgenic for the SCL and LMO1 genes. Cells from the primary malignancies initially grew very slowly in vitro, loosely attached to a stromal layer, before gaining the ability to proliferate independently. Upon gaining the ability to proliferate in the absence of a stromal layer, these cell lines grew rapidly, doubling every 14-23 h, to a very high density, approaching 10(7) cells/ml. Whereas the tumors which arise in SCL/LMO1 double transgenic mice are typically diploid or pseudodiploid, the cell lines were all grossly aneuploid, suggesting the possibility that additional genetic events were selected for in vitro. Given that SCL and LMO1 gene activation are both commonly seen in human patients with T cell acute lymphoblastic leukemia, these cell lines may be a useful in vitro model for the human disease.

Disordered T-cell development and T-cell malignancies in SCL LMO1 double-transgenic mice: parallels with E2A-deficient mice.

The gene most commonly activated by chromosomal rearrangements in patients with T-cell acute lymphoblastic leukemia (T-ALL) is SCL/tal. In collaboration with LMO1 or LMO2, the thymic expression of SCL/tal leads to T-ALL at a young age with a high degree of penetrance in transgenic mice. We now show that SCL LMO1 double-transgenic mice display thymocyte developmental abnormalities in terms of proliferation, apoptosis, clonality, and immunophenotype prior to the onset of a frank malignancy. At 4 weeks of age, thymocytes from SCL LMO1 mice show 70% fewer total thymocytes, with increased rates of both proliferation and apoptosis, than control thymocytes. At this age, a clonal population of thymocytes begins to populate the thymus, as evidenced by oligoclonal T-cell-receptor gene rearrangements. Also, there is a dramatic increase in immature CD44(+) CD25(-) cells, a decrease in the more mature CD4(+) CD8(+) cells, and development of an abnormal CD44(+) CD8(+) population. An identical pattern of premalignant changes is seen with either a full-length SCL protein or an amino-terminal truncated protein which lacks the SCL transactivation domain, demonstrating that the amino-terminal portion of SCL is not important for leukemogenesis. Lastly, we show that the T-ALL which develop in the SCL LMO1 mice are strikingly similar to those which develop in E2A null mice, supporting the hypothesis that SCL exerts its oncogenic action through a functional inactivation of E proteins.

Association of germline p53 mutation with MLL segmental jumping translocation in treatment-related leukemia.

Segmental jumping translocations are chromosomal abnormalities in treatment-related leukemias characterized by multiple copies of the ABL and/or MLL oncogenes dispersed throughout the genome and extrachromosomally. Because gene amplification potential accompanies loss of wild-type p53, we examined the p53 gene in a case of treatment-related acute myeloid leukemia (t-AML) with MLL segmental jumping translocation. The child was diagnosed with ganglioneuroma and embryonal rhabdomyosarcoma (ERMS) at 2 years of age. Therapy for ERMS included alkylating agents, DNA topoisomerase I and DNA topoisomerase II inhibitors, and local radiation. t-AML was diagnosed at 4 years of age. The complex karyotype of the t-AML showed structural and numerical abnormalities. Fluorescence in situ hybridization analysis showed multiple copies of the MLL gene, consistent with segmental jumping translocation. A genomic region including CD3, MLL, and a segment of band 11q24 was unrearranged and amplified by Southern blot analysis. There was no family history of a cancer predisposing syndrome, but single-strand conformation polymorphism (SSCP) analysis detected identical band shifts in the leukemia, ganglioneuroma, ERMS, and normal tissues, consistent with a germline p53 mutation, and there was loss of heterozygosity in the ERMS and the t-AML. Sequencing showed a CGA-->TGA nonsense mutation at codon 306 in exon 8. The results of this analysis indicate that loss of wild-type p53 may be associated with genomic instability after DNA-damaging chemotherapy and radiation, manifest as a complex karyotype and gene amplification in some cases of t-AML.

DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.

An scl gene product lacking the transactivation domain induces bony abnormalities and cooperates with LMO1 to generate T-cell malignancies in transgenic mice.

The product of the scl (also called tal-1 or TCL5) gene is a basic domain, helix-loop-helix (bHLH) transcription factor required for the development of hematopoietic cells. Additionally, scl gene disruption and dysregulation, by either chromosomal translocations or a site-specific interstitial deletion whereby 5' regulatory elements of the sil gene become juxtaposed to the body of the scl gene, is associated with T-cell acute lymphoblastic leukemia (ALL) and T-cell lymphoblastic lymphoma. Here we show that an inappropriately expressed scl protein, driven by sil regulatory elements, can cause aggressive T-cell malignancies in collaboration with a misexpressed LMO1 protein, thus recapitulating the situation seen in a subset of human T-cell ALL. Moreover, we show that inappropriately expressed scl can interfere with the development of other tissues derived from mesoderm. Lastly, we show that an scl construct lacking the scl transactivation domain collaborates with misexpressed LMO1, demonstrating that the scl transactivation domain is dispensable for oncogenesis, and supporting the hypothesis that the scl gene product exerts its oncogenic action through a dominant-negative mechanism.

Topoisomerase II inhibitors induce DNA double-strand breaks at a specific site within the AML1 locus.

Treatment-related acute myeloid leukemia (t-AML) following successful therapy of a primary malignancy has been recognized with increasing frequency among cancer survivors over the past several years. Many of these t-AML cases are associated with the use of intensive chemotherapy regimens that employ one or more agents which target eukaryotic topoisomerase II (topo II), and demonstrate non-random chromosomal translocations involving either the MLL (ALL-1, HRX) gene at 11q23 or the AML1 gene at 21q22. Although many investigators have speculated that these translocations are induced by the therapeutic use of topo II inhibitors, the molecular sequence of events by which topo II inhibitors might induce a chromosomal translocation are not well understood. We describe here the reproducible induction of highly specific, double-strand DNA cleavage at a specific site within the AML1 locus by topo II inhibitors. This DNA cleavage, which maps to a region of the AML1 locus frequently disrupted by chromosomal translocations, can be induced in several cell lines, with multiple different topo II inhibitors, indicating that this phenomenon is not restricted to a specific cell type or specific topo II inhibitor. It is conceivable that site-specific double-strand DNA cleavage within the AML1 locus induced by topo II inhibitors represents the initial molecular event leading to a chromosomal translocation and t-AML.

Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors.

The MLL gene located at 11q23 is frequently disrupted by chromosomal translocation in a wide spectrum of newly diagnosed acute leukemias. Recently, it has become apparent that the MLL gene is very frequently disrupted by chromosomal translocations in patients with secondary leukemias associated with chemotherapeutic regimens incorporating topoisomerase II inhibitors. These secondary leukemias associated with topoisomerase II inhibitors (most commonly teniposide, etoposide, or doxorubicin) have distinct clinical and biologic features which have led to the speculation that they are induced by treatment with topoisomerase II inhibitors. We have identified a site within the MLL breakpoint cluster region (bcr) that is highly sensitive to double-strand DNA cleavage induced by topoisomerase II inhibitors. This finding is quite specific and highly reproducible. Although it was initially discovered in malignant lymphoblasts isolated from a patient receiving multiagent chemotherapy, this site-specific double-strand DNA cleavage can be induced in tissue culture using malignant cell lines as well as peripheral blood from normal individuals. Site-specific cleavage occurs in a significant fraction of cells using a variety of model systems, is both time and dose dependent, and can be induced with either doxorubicin or etoposide. This site-specific cleavage maps to the same region as a consensus topoisomerase II cleavage site within the MLL bcr. These results suggest that site specific cleavage within the MLL bcr induced by topoisomerase II inhibitors may be an early step leading to MLL translocations and secondary leukemia.

Complex MLL rearrangement in a patient with T-cell acute lymphoblastic leukemia.

MLL (also known as ALL-I, HTRX, or HRX) gene translocations are among the most common chromosomal abnormalities recognized in both B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). However, MLL gene rearrangements are uncommon in T-cell ALL. We recently detected an MLL gene rearrangement in a patient with typical T-cell ALL. We recently detected an MLL gene rearrangement in a patient with typical T-cell ALL (CD2+, CD4+, CD5+, CD7+, CD8+, HLA DR-) and an apparently normal karyotype (46,XX). The rearrangement was cloned and characterized; a DNA fragment distal to the breakpoint was mapped by fluorescence in situ hybridization (FISH) to 19p13, indicating that the leukemic blasts had undergone a cytogenetically undetected rearrangement involving chromosomes 11 and 19. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated an in-frame fusion mRNA between the amino terminus of MLL and the carboxy terminus of ENL (also known as MLLT1 or LTG19), a gene that has been mapped to 19p13. In addition, MLL sequences distal (telomeric) to the breakpoint were deleted from the genome, which precludes the formation of a reciprocal ENL/MLL fusion protein. These findings suggest that an MLL/ENL fusion protein (and not a reciprocal ENL/MLL fusion) was likely to be pathogenic in this patient, and they reinforce previous studies showing that leukemic blasts with apparently normal karyotype may harbor MLL rearrangements. Additionally, this report provides the first conclusive evidence of an MLL/ENL gene fusion characterized at a molecular level in a patient with T-cell ALL.

Cloning and characterization of TCTA, a gene located at the site of a t(1;3) translocation.

We have cloned and characterized a novel gene at the site of a t(1;3)(p34;p21) translocation breakpoint in T-cell acute lymphoblastic leukemia. A cDNA for this gene, for which we propose the designation TCTA (T-cell leukemia translocation-associated gene), has been cloned. TCTA mRNA is expressed ubiquitously in normal tissues, with the highest levels of expression seen in the kidney. The TCTA gene is conserved throughout evolution in organisms ranging from Drosophila to humans. A short open reading frame encodes a predicted M(r) 12,000 protein without strong homology to any previously reported proteins. Of note, genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines tested, suggesting loss of one of the two copies of the gene.

Concurrent presence of inv(14)(q11q32) and t(4;11)(q21;q23) in pre-B acute lymphoblastic leukemia.

The inv(14)(q11q32) is a non-random chromosomal aberration which has been associated with a variety of T-cell malignancies. We have studied a case of inv(14)(q11q32) that is unique in several respects. First, the inversion, which is expressed at the mRNA level, occurred in the context of a pre-B acute lymphoblastic leukemia (ALL) as opposed to a T-cell malignancy. Second, cloning and sequencing of the inversion revealed that it resulted from a fusion between an immunoglobulin heavy chain variable (V) segment and a T-cell receptor delta diversity (D) segment. In addition, the patient had a second chromosomal abnormality at diagnosis, a t(4;11)(q21;q23) which disrupted the MLL gene. The fact that there were two distinct chromosomal abnormalities at diagnosis enabled us to address the question of leukemic clonal evolution during the course of this patient's disease. We present evidence suggesting that the t(4;11)(q21;q23) occurred first, with the inv(14)(q11q32) occurring as a second event.

Reversal of C1300 murine neuroblastoma multidrug resistance by cremophorEL, a solvent for cyclosporin A.

We previously developed a homoharringtonine resistant C-1300 neuroblastoma cell line with cross-resistance to adriamycin and increased levels of p-glycoprotein, and showed that drug resistance could be reversed in this cell line by cyclosporin A. The present study shows that cremophor EL, a parenteral vehicle for cyclosporin A, can also completely reverse this multidrug resistance in a clonogenic assay system. Cremophor EL incubated with resistant cells for up to six days did not reduce levels of p-glycoprotein. Intracellular homoharringtonine analysis using HPLC revealed increased drug accumulation in resistant cells treated with cremophor EL. The increased drug level was not due to blocking of drug efflux commonly seen in other multidrug resistant models. The data suggest that resistance modulation with cyclosporin A should be interpreted with caution when cremophor EL is a solvent. Our work suggests cremophor EL, a relatively nontoxic lipophylic solvent, may have a direct effect on membrane permeability, although other mechanisms cannot be ruled out.

Cremophor-EL enhances taxol efficacy in a multi-drug resistant C1300 neuroblastoma cell line.

We have previously developed a homoharringtonine (HHT) resistant murine C1300 neuroblastoma cell line with increased p-glycoprotein expression and cross resistance to Adriamycin. Drug resistance in this cell line was reversed using cyclosporin-A, dipyridamole and cremophor-EL (CRE). Because of the high CRE content of parenteral taxol, we examined the ability of this solvent to reverse taxol cross-resistance in this cell line. Comparative ID-50s using clonogenic assays in agar indicate a 214-fold resistance to HHT. CRE reverses taxol cross-resistance in a dose-dependent manner from 0.003 to 0.1%, and is maximally effective at a subtoxic concentration of 0.03%. High pressure liquid chromatography (HPLC) analysis of taxol treated C1300/HHT cells reveal that CRE causes changes in intracellular drug levels that are not related to drug efflux. Our work shows that clinical preparations of taxol, when diluted to effective doses, contain enough CRE to mitigate multi-drug resistance. Clinical successes of taxol in refractory tumors may be due in part to the ability of its CRE base to reverse multi-drug resistance.

Modulation of drug resistance in homoharringtonine-resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.

In vitro ultrastructural localization of the catalytic activity of adenosine deaminase in murine C-1300 neuroblastoma.

Adenosine deaminase (ADA) activity was localized at the surface membrane of the mouse C-1300 neuroblastoma by incubation of a confluent tissue culture monolayer grown on Lux-Permanox cultureware with 6-chloropurine ribonucleoside (CPR). This substrate is dechlorinated by ADA to form Cl-. At loci of ADA activity, Cl- is precipitated with added silver ion (Ag+), and electron dense metallic silver (Ag degree) is formed upon exposure to light. The incubation was conducted in 0.2 M HEPES buffer (277 mOs) at 37 degrees C, pH 7.4, which contained 1 mM CPR, for 5 min (in this buffer, this is four times the Km); the control lacked the substrate. After completion of the incubation, the monolayer was briefly rinsed with 0.2 M HEPES and 2.5% glutaraldehyde in 0.2 M HEPES containing AgNO3 at a final concentration of 2 mM. Dehydration was accomplished in a graded series of ethanol followed by embedment in the L. R. White resin at 60 degrees C overnight. Thin sections (80 to 100 mm), cut parallel to the monolayer, showed ADA activity at the cells' surface membrane with a smaller amount of activity evenly distributed in the subadjacent ectoplasmic zone. The control lacked any silver grain localization. Since in preliminary studies of neuronal tissue the ADA activity was minimal, these findings may contribute to developing a diagnostic cancer screening test.

The significance of the ratio of blood to marrow hematopoietic progenitors (CFU-GM and CFU-GEMM) in relation to disease state of patients with acute lymphoblastic leukemia.

We studied a total of 143 patients with 231 assays of CFU-GM, of these, 45 patients were studied with 58 simultaneous assays of CFU-GEMM in an attempt to correlate progenitor colony growth with disease state. Blood and marrow leukocytes were cultured in a standard methylcellulose culture system using normal human leukocyte colony stimulating activity. Results are expressed as natural logarithm of the ratio of blood colonies to marrow colonies. A high ratio of blood to marrow (greater than 1) was seen in active disease while a low ratio occurred during remission. Eighty-seven per cent of CFU-GM matched pair samples with active disease had high ratios while less than 1% of remission samples were high. In addition, patients with suspected relapse or relapse up to 14 months after this test often had high ratios indicating this test could be of prognostic value. It is not known if this effect of ALL is unique to ALL or if it occurs as a reaction to any hematopoietic stress.

In vitro production of granulocyte-macrophage colony-stimulating activity by murine bone marrow and spleen following vinblastine administration in vivo.

The production of granulocyte-macrophage colony-stimulating activity (CSA) by isolated murine femur shafts, non-dispersed bone marrow and spleens was assessed following administration of Vinblastine (VLB). These organs were removed from 2 h to 10 days post-VLB and allowed to condition endotoxin-free medium for 48 h. CSA activity was assessed using a soft agar cloning system. The data demonstrate that CSA elaboration was maximal 24 h post-VLB, corresponding to the nadir of bone marrow GM-CFC, and 6 h after splenic reached a minimum. No relationship between peripheral blood granulocyte count or serum endotoxin levels were observed.

The role of eosinophil in regulation of granulopoiesis.

An association between eosinophilia and neutropenia has been observed in a number of clinical conditions. To probe the role of eosinophils in granulopoiesis, marrow and peritoneal eosinophils, obtained from Schistosoma mansoni-infected mice, were separated and purified. Normal bone marrow cells were cultured in semi-solid culture medium in concentrations ranging from 5 X 10(4) to 5 X 10(5), with and without added eosinophils. To examine whether high prostaglandin E (PGE) content of eosinophils affects granulopoiesis, indomethacin was added to duplicate marrow cultures containing eosinophils. The addition of eosinophils to normal syngeneic marrow culture caused a significant inhibition of granulocyte-macrophage colony formation (CFU-GM) in culture. This suppressive effect was reversible upon addition of indomethacin. These findings suggest that eosinophils, in vitro, are capable of inhibition of granulopoiesis. The reversal of this effect by indomethacin indicates that this suppression may be prostaglandin mediated.

Human T-cell leukemia-associated cell surface glycoprotein GP37: studies with three monoclonal antibodies and a rabbit antiserum.

Three monoclonal antibodies (mAbs), termed SN2, SN2a and SN2b, were used in the present work to study a human T-cell leukemia-associated cell surface glycoprotein, GP37. Strong specificity of mAbs SN2, SN2a and SN2b for T leukemia cells was demonstrated by radioimmunoassay and fluorescence-activated cell sorter (FACS) analysis. GP37 was not detected on normal human peripheral blood lymphocytes, purified normal T-cells, normal thymocytes nor normal bone marrow cells. Furthermore, GP37 was barely detectable on phytohemagglutinin (PHA)- and Concanavalin A (Con A)-activated T-cells. The results indicate clinical utility of these mAbs. Competitive binding experiments show that the epitopes recognized by SN2 and SN2a are sufficiently close to each other to allow complete reciprocal inhibition of binding whereas the epitopes recognized by SN2 and SN2b are less close to allow only partial reciprocal binding inhibition. The biochemical nature of antigenic determinants defined by these mAbs was studied by treating T leukemia cells with trypsin, chymotrypsin, thermolysin, neuraminidase and mixed glycosidases. The results suggest that the antigenic determinants defined by these mAbs all consist of the protein moiety of the glycoprotein GP37. No significant antigenic modulation was observed when T leukemia cells were reacted with SN2. In a sequential immunoprecipitation experiment, a 125I-labeled leukemia antigen preparation was first treated with a rabbit anti-T leukemia antiserum. The latter had been prepared by immunizing a rabbit with a partially purified human T leukemia antigen preparation and showed a good specificity for T leukemia cells. Subsequent treatment of the labeled antigen preparation with SN2 showed that SN2 antigen had been precleared. Thus, both mouse mAb SN2 and the rabbit anti-T leukemia antiserum react with the same GP37 molecule.

The effect of vinblastine on CFU-E in normal and W anaemic mice.

Using a plasma clot technique, we studied the comparative levels of in vitro erythroid colony forming units (CFU-E) of normal and W anaemic mice following intravenous (IV) injection of 6.25 mg/kg of vinblastine (VBL). Mice used in this study were C57Bl/6J, WBB6F1, (+/+,Wv/+ and W/Wv). CFU-E content was looked at on day 0, 1, 3, 5, 7 and 10 following IV VBL. The CFU-E content of bone marrow (BM) and spleen was adequate prior to VBL treatment in all four mouse strains tested. We found IV VBL resulted in a significant rebound of CFU-E production in spleens of all four mouse strains. Most CFU-E production occurred on days 7 and 10 post VBL. The total CFU-E content of BM was not significantly affected by VBL in C57Bl/6J, WBB6F1, +/+ and Wv/+ mice. However, a significant reduction of total CFU-E was seen in the BM of WBB6F1, W/Wv mice. This plus the fact that increased CFU-E is seen in the spleen of all mouse strains including W/Wv suggests that there may be differing cellular or environmental factor(s) in different hematological organs controlling CFU-E of the W/Wv mouse.