Thymic overexpression of Ttg-1 in transgenic mice results in T-cell acute lymphoblastic leukemia/lymphoma.

T-cell translocation gene 1 (Ttg-1), also called rhombotin, is deregulated upon translocation into the alpha/delta T-cell receptor loci in acute lymphoblastic leukemias bearing the t(11;14)(p15;q11). Ttg-1 encodes a nuclear protein, expressed predominantly in neuronal cells, which belongs to a novel family of transcription factors possessing LIM domains. We utilized the lck proximal promoter to overexpress this candidate oncogene in immature thymocytes of transgenic mice. lckPr Ttg-1 mice develop immature, aggressive T-cell leukemia/lymphomas. Tumor incidence is proportional to the level of Ttg-1 expression. Most tumors contain CD4+8+ cells as well as CD4-8+ cells, which have an immature rather than a mature peripheral phenotype. Ttg-1-induced tumorigenesis preferentially affects a minority population of thymocytes representing an immature CD4-8+ intermediate stage between double-negative CD4-8- cells and double-positive CD4+8+ cells. This model indicates that the aberrant expression of putative transcription factors plays a primary role in the genesis of T-cell acute lymphoblastic leukemias.

The t(11;14)(p15;q11) in a T-cell acute lymphoblastic leukemia cell line activates multiple transcripts, including Ttg-1, a gene encoding a potential zinc finger protein.

Interchromosomal translocations within lymphoid neoplasms frequently involve the antigen receptor genes. We cloned the breakpoints of the t(11;14)(p15;q11) in a CD3-negative T-cell acute lymphoblastic leukemia cell line (RPMI 8402) in order to identify new genes potentially involved in T-cell neoplasia. An extensive comparison of both breakpoints and their germ line counterparts indicated that an inadvertant recombinase-mediated break at chromosome segment 11p15 recombined with the delta T-cell receptor at 14q11. The derivative 11 breakpoint resembles a coding joint in which 11p15 rather than a variable region was introduced 5' to a D delta 1 D delta 2 J delta 1 intermediate rearrangement. Conversely, the derivative 14 breakpoint corresponds to a signal joint between the 5' heptamer-spacer-nonamer recombinational signal of D delta 1 and an isolated heptamer at 11p15. Multiple, apparently distinct transcripts were found flanking both breakpoints of 8402. RNAs of 3.5, 4.4, 1.4, and 8.0 kilobases originating from either side of the derivative 14 breakpoint were highly expressed in 8402 compared with other cells. This suggests that this translocation deregulated multiple genes and provides the opportunity to assess any multifactorial contribution they may have to malignancy. We cloned and sequenced several cDNAs representing the 1.4-kilobase transcript (termed Ttg-1 [T-cell translocation gene 1]) from an 8402 library. The predicted protein of 156 amino acids contained two internal repeats which could potentially form zinc fingers.

Potentiation of lymphomagenesis by methylnitrosourea in mice transgenic for LMO1 is blocked by O6-alkylguanine DNA-alkyltransferase.

We evaluated induction of lymphomas by the methylating carcinogen, N-methylnitrosourea [MNU], in transgenic mice expressing both LMO1 and the DNA repair gene, MGMT, in the thymus. The goal was to determine whether environmental mutagens shorten the latency or increase the incidence of LMO1 + lymphomas and whether mice transgenic for both LMO1 and MGMT, and thereby able to repair O6-methylguanine DNA adducts induced by MNU, would be protected. Mice heterozygous for LMO1 or MGMT were crossed and offspring treated with MNU at 6 weeks of age. MNU induced lymphoma incidence was highest in the LMO1 mice, 91% and lowest in the hMGMT + mice, 15%. MNU induced K-ras mutations in codon 12 in non-MGMT transgenics resulted in a shorter latency of tumors and accounting for half of the early lymphomas in LMO1 mice. The effect of MNU was abrogated in the LMO1/hMGMT transgenic mice, indicating the ability of MGMT expression to block the carcinogenic effect of MNU even in cancer prone mice. Thus, methylating agents potentiate lymphomagenesis of LMO1, in part through activation of K-ras and the MAPK pathway, a process which appear to synergize with LMO1 mediated transcription activation. O6-alkylguanine DNA-alkyltransferase mediated DNA repair effectively blocks chemical carcinogenesis in mice carrying the LMO1 oncogene.

Kinetics of native insulin in diabetic, obese, and aged men.

The kinetics of unlabeled porcine insulin were studied in 69 nondiabetic male subjects aged 18-83 yr with obesity indexes of 0.93 - 1.51 and in 12 maturity-onset diabetics age 46-78 yr with obesity indexes of 0.95-1.56 by using the euglycemic clamp technique. Analysis of the insulin kinetic data by using a mathematical model permitted the determination, for each individual, of steady state distribution masses and degradation rate constants. The individuals were grouped to allow comparison of the results on the basis of age, obesity index, or diabetes. The responses over a period of 120 min to an infusion and wash out of insulin show some transient as well as steady state differences with age, obesity, or diabetes. Analysis of these data by use of compartmental models leads to the conclusion that in the steady state the ratio of insulin in extravascular spaces to that in plasma (T/P) is decreased in the moderately obese group (26%) and in the diabetic group (17%) but increased in the older group (13%) when each is compared with the appropriate control. Since extravascular insulin includes both insulin bound to receptors and insulin in the interstitial fluid, the observed changes in the extravascular to plasma mass ratio most likely reflect changes in in vivo binding to receptors, although the magnitude of the change would be modified somewhat by changes in the size of the interstitial spaces relative to plasma. In addition, the rate of entry of new insulin into plasma (BSDR) was increased in the diabetic population (45%; P less than 0.02) as well as in the moderately obese group (27%) but was decreased somewhat in the older group (11%). The following general conclusions can be drawn from the results: The pattern of parameter changes seen with obesity is similar to that seen with maturity-onset diabetes. The decrease in T/P seen with obesity and with maturity-onset diabetes cannot be accounted for solely by changes in fasting plasma insulin levels in these populations. The pattern of changes seen in the older subjects is opposite that seen in the maturity-onset diabetics, which suggests that diabetes is a perturbation distinct from the normal aging process. Finally, the changes in the metabolism of insulin are not large, making it unlikely that they are the sole cause of the major alterations in glucose tolerance seen with aging, obesity, or diabetes.

Aging and ethanol metabolism.

The effect of aging on the distribution and elimination of ethanol was studied in a group of 50 healthy subjects ranging in age from 21 to 81 yr (mean, 53.3). Ethanol was administered in a continuous 1-hr infusion at a mean rate of 375 mg/m2 body surface area/min (equivalent to a mean dose of 0.57 gm/kg body weight). Serial blood samples for the determination of ethanol concentration was obtained at 15- to 30-min intervals for up to 4 hr post infusion. Ethanol elimination and distribution were evaluated with the aid of a two-compartment model. Rates of ethanol elimination were not affected by age. Peak ethanol concentration in blood water at the end of the infusion period was correlated with age (r= 0.55, p less than 0.001). Lean body mass and total volume of distirbution fo the ethanol were negatively correlated with age. The smaller volume of distirbution, in association with the decreased lean body mass, most likely explains the higher peak ethanol concentration found in the blood after administration of an ethanol does on the basis of surface area in the old as compared with the young subjects. This study demonstrates that age-related changes in body composition are important factors in the study of ethanol metabolism and its pharmacologic effects.

Activation of heparin cofactor II by fibroblasts and vascular smooth muscle cells.

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated by dermatan sulfate, heparan sulfate, and heparin. Purified HCII or defibrinated plasma was incubated with washed confluent cell monolayers, 125I-thrombin was added, and the rate of formation of covalent 125I-thrombin-inhibitor complexes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fibroblasts and porcine aortic smooth muscle cells accelerated inhibition of thrombin by HCII 2.3-7.5-fold but had no effect on other thrombin inhibitors in plasma. Human umbilical vein endothelial cells and mouse macrophage-derived cells did not accelerate the thrombin-HCII reaction. IMR-90 normal human fetal lung fibroblasts treated with heparinase or heparitinase accelerated the thrombin-HCII reaction to the same degree as untreated cells. In contrast, treatment with chondroitinase ABC almost totally abolished the ability of these cells to activate HCII while chondroitinase AC had little or no effect, suggesting that dermatan sulfate was responsible for the activity observed. [35S]Sulfate-labeled proteoglycans were isolated from IMR-90 fibroblast monolayers and conditioned medium and fractionated into two peaks on Sepharose CL-2B. The lower Mr proteoglycans contained 74-76% dermatan sulfate and were 11-25 times more active with HCII than the higher Mr proteoglycans which contained 68-97% heparan sulfate. The activity of the lower Mr proteoglycans decreased 70-90% by degradation of the dermatan sulfate component with chondroitinase ABC. These results confirm that dermatan sulfate proteoglycans are primarily responsible for activation of HCII by IMR-90 fibroblasts. We suggest that HCII may inhibit thrombin when plasma is exposed to vascular smooth muscle cells or fibroblasts.

Nutritional edema in a rat model of protein deficiency: significance of the dietary potassium and sodium content.

The importance of the levels of dietary sodium and potassium in the etiology of gross nutritional edema in a rat model was explored. For this purpose a mineral mixture (EAM) was designed to permit changing the levels of sodium and potassium while maintaining other components of the mixture. The mixture supplied (as % of diet) 0.075 sodium and 0.270 potassium. In study 1 the EAM mixture was found to support adequate growth (approximately 6 g/d) in well-nourished rats. In study 2 the effects of feeding the diet in either agar gel or dry form were explored. The agar gel diet did not aggravate disturbances in body water balance in rats receiving low protein (0.75 and 1.0% lactalbumin) diets for 20 wk. In study 3 the effects of changes in the sodium and potassium content were evaluated with respect to development of edema and body composition. Excessive levels of sodium or potassium (each 493 mg/100 g diet) in the low protein diet (0.5% lactalbumin) increased mortality and the prevalence of gross edema. When dietary sodium and potassium were closer to the estimated requirement for the rat (0.075% and 0.270%, respectively) there was no development of visible edema in protein-restricted rats. Measurements of exchangeable body sodium, total body water and extracellular and intracellular fluid spaces in the animals indicated that fluid and electrolyte changes result largely from dietary protein restriction alone. However, these changes only proceed to a condition of visible edema where an excessive or unbalanced intake of sodium and potassium is superimposed upon protein deficiency.

The biology and treatment of acute lymphoblastic leukemia in adults.

Despite reports to the contrary, only a small minority of adults with ALL are currently cured. Results have improved modestly with more intensive postremission chemotherapy and with tailoring of protocols in individuals with specific subsets of ALL. The use of growth factors may further improve treatment results. The performance of allogeneic BMT in first remission is clearly effective in some individuals, eg, those with Ph1-positive ALL, but it is unclear whether it is advantageous in most individuals. There are little data supporting the effectiveness of autotransplantation, as currently performed in ALL, despite its theoretical potential. Advances in understanding the biology of ALL have led to new approaches currently under basic and clinical investigation. These include serial studies of minimal residual disease by a variety of techniques to tailor treatment, the development of conjugated MoAbs to lymphoid cell antigens and immunologic and biochemical approaches to chimeric RNA and peptides generated by abnormal fusion genes. It seems likely that substantial improvement in the treatment of adult ALL awaits better characterization of the biology of this disease. However, some improvement will occur through empirical clinical research. It is critical that physicians recognize the poor results with current therapeutic approaches and enter patients into large well-designed clinical trials.

T-cell translocation gene 1 (Ttg-1) encodes a nuclear protein normally expressed in neural lineage cells.

We previously identified and cloned T-cell translocation gene 1 (Ttg-1), a putative zinc finger protein, as a result of its deregulated expression in a T-cell acute lymphoblastic leukemia cell line (RPMI 8402) with a t(11;14)(p15;q11). We have now characterized its genomic organization and identified the major transcriptional start site to lie within an initiator-like motif. Ttg-1 is normally expressed in mouse brain and not in thymus. The mouse neuroblastoma cell line, N2a, also expresses Ttg-1. Antibodies raised against a TrpE-Ttg-1 fusion protein precipitate an 18-Kd nuclear protein from metabolically labeled 8402 cells. Immunofluorescence of N2a cells shows a nuclear pattern. The two potential zinc finger domains in Ttg-1 are highly homologous to similar regions in lin-11, mec-3, and lsl-1. This data suggests that Ttg-1 may be involved in gene regulation.

Human delta T cell receptor: rearrangement, deletion, and translocation.

Individual T cells express the CD3 molecule in association with alternative gamma delta or alpha beta heterodimeric T cell receptors (TCR). The delta TCR, used in one type of T cell is located within the alpha TCR gene used in quite another cell. T cell precursors and occasional gamma delta T cells in humans possess an unexpected 2.0 Kb mRNA in which a tandemly repeated motif, T early alpha (TEA), has been spliced to the constant (C alpha) region. Long range pulse field gel mapping as well as molecular cloning reveal that TEA is located immediately 5' to the most upstream joining (J alpha) segments of the alpha TCR locus. The human delta TCR locus is immediately 5' to TEA and diversity (D delta 1 +2), J delta 1 +2, C delta, and TEA are linked within 35 Kb. The human delta TCR locus conserves a 12/23 bp spacer paradigm and D delta 1 and D delta 2 are frequently recombined as D delta 1/D delta 2, and reveal exonucleolytic trimming with extensive "N" segment addition. Thus, despite the predominant use of one V delta and J delta segment considerable delta diversity is generated. T cell acute lymphoblastic leukemias (ALL) represent clonal expansions of maturationally arrested cells at specific stages of thymic ontogeny. The gamma delta T-ALLs display allelic exclusion for the delta TCR. Moreover, pre T cells within this group indicate that delta TCR rearrangement can occur prior to the activation of gamma, beta, and alpha loci.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of arterial versus venous sampling on analysis of glucose kinetics in man.

A compartmental model is presented to account for transient and steady-state changes in blood glucose concentration which result from transit through the forearm and hand in man. This model permits the inter-conversion of arterial and venous data and the derivation of arterial equivalent total body glucose models from venous data. Data were obtained from subjects in the basal state following a pulse injection of [1-14C]glucose tracer. An artery, an antecubital vein, and a dorsal vein of a heated hand (68 degrees C environment) were sampled. Blood transit time is shorter 0.3 vs. 1.0 min) and irreversible glucose loss is reduced (1.9 vs. 2.9%) in the heated hand preparation when compared to the antecubital vein preparation. Because of the smaller correction required and the smaller variation among individuals when heated hand rather than antecubital vein data are obtained, we suggest that for analysis of whole-body kinetics such data should be used along with the compartmental model correction when arterial data cannot be obtained.

Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain.

Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis.

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.

The t(10;14)(q24;q11) of T-cell acute lymphoblastic leukemia juxtaposes the delta T-cell receptor with TCL3, a conserved and activated locus at 10q24.

We cloned the t(10;14) recurrent translocation from CD3-negative T-cell acute lymphoblastic leukemia cells. The breakpoint at 14q11 involved an intermediate rearrangement of the delta T-cell receptor locus, suggesting that the translocation arose at the time of antigen receptor assemblage. Translocation introduced chromosome segment 10q24 as proven by hybridization of a breakpoint-derived probe to flow-sorted chromosomes and metaphase chromosomes. Two t(10;14) breakpoints were clustered within a 600-base-pair region of 10q24 but no heptamer-spacer-nonamer motifs resembling T-cell receptor/immunoglobulin rearrangement signals were noted at the breakpoint. A locus distinct from terminal deoxynucleotidyltransferase was found at 10q24. Evolutionarily conserved regions surrounding the 10q24 breakpoint were examined for transcriptional activity. A region telomeric to the 10q24 breakpoint, expected to translocate to the der(14) chromosome, recognized an abundant 2.9-kilobase RNA in a t(10;14) T-cell leukemia. This locus was not active in a variety of other normal and neoplastic T cells, arguing that it was deregulated by the introduction of the T-cell receptor. This locus is a candidate for a putative protooncogene, TCL3, involved in T-cell neoplasia.