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The huge exopeptidase, tripeptidyl-peptidase II (TPP II), appears to be involved in a large number of important biological processes. It is present in the cytosol of most eukaryotic cells, where it removes tripeptides from free amino termini of longer peptides through a 'molecular ruler mechanism'. Its main role appears to be general protein degradation, together with the proteasome. The activity is increased by stress, such as during starvation and muscle wasting, and in tumour cells. Overexpression of TPP II leads to accelerated cell growth, genetic instability and resistance to apoptosis, whereas inhibition or down-regulation of TPP II renders cells sensitive to apoptosis. Although it seems that humans can survive without TPP II, it is not without consequences. Recently, patients with loss-of-function mutations in the TPP2 gene have been identified. They suffer from autoimmunity leading to leukopenia and other consequences. Furthermore, a missense mutation in the TPP2 gene is associated with a sterile brain inflammation condition mimicking multiple sclerosis. This review will summarise what is known today regarding the activity and structure of this very large enzyme complex, and its potential function in various cellular processes. It is clear that more research is needed to identify natural substrates and/or interaction partners of TPP II, which can explain the observed effects in different cellular contexts., (Copyright © 2019 The Author. Published by Elsevier B.V. All rights reserved.)

Objective: To ascertain the genetic cause of a consanguineous family from Syria suffering from a sterile brain inflammation mimicking a mild nonprogressive form of MS., Methods: We used homozygosity mapping and next-generation sequencing to detect the disease-causing gene in the affected siblings. In addition, we performed RNA and protein expression studies, enzymatic activity assays, immunohistochemistry, and targeted sequencing of further MS cases from Austria, Germany, Canada and Jordan., Results: In this study, we describe the identification of a homozygous missense mutation (c.82T>G, p.Cys28Gly) in the tripeptidyl peptidase II ( TPP2 ) gene in all 3 affected siblings of the family. Sequencing of all TPP2 -coding exons in 826 MS cases identified one further homozygous missense variant (c.2027C>T, p.Thr676Ile) in a Jordanian MS patient. TPP2 protein expression in whole blood was reduced in the affected siblings. In contrast, TPP2 protein expression in postmortem brain tissue from MS patients without TPP2 mutations was highly upregulated., Conclusions: The homozygous TPP2 mutation (p.Cys28Gly) is likely responsible for the inflammation phenotype in this family. TPP2 is an ubiquitously expressed serine peptidase that removes tripeptides from the N-terminal end of longer peptides. TPP2 is involved in various biological processes including the destruction of major histocompatibility complex Class I epitopes. Recessive loss-of-function mutations in TPP2 were described in patients with Evans syndrome, a rare autoimmune disease affecting the hematopoietic system. Based on the gene expression results in our MS autopsy brain samples, we further suggest that TPP2 may play a broader role in the inflammatory process in MS.

Introduction: Antigen-specific immunotherapy could provide a targeted approach for the treatment of multiple sclerosis that removes the need for broad-acting immunomodulatory drugs. ATX-MS-1467 is a mixture of four peptides identified as the main immune-dominant disease-associated T-cell epitopes in myelin basic protein (MBP), an autoimmune target for activated autoreactive T cells in multiple sclerosis. Previous animal studies have shown that ATX-MS-1467 treatment prevented the worsening of signs of disease in experimental autoimmune encephalitis (EAE) in the humanized (DR2 × Ob1)F1 mouse in a dose-dependent fashion., Methods and Results: Our study extends these observations to show that subcutaneous treatment with 100 µg of ATX-MS-1467 after induction of EAE in the same mouse model reversed established clinical disability (p < 0.0001) and histological markers of inflammation and demyelination (p < 0.001) compared with vehicle-treated animals; furthermore, in longitudinal magnetic resonance imaging analyses, disruption of blood-brain barrier integrity was reversed, compared with vehicle-treated animals (p < 0.05). Chronic treatment with ATX-MS-1467 was associated with an enduring shift from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in interleukin 10 secretion, relative to interleukin 2, interleukin 17 and interferon γ, a decrease in splenocyte proliferation and an increase in interleukin 10 + Foxp3 - T cells in the spleen., Conclusion: Our results suggest that ATX-MS-1467 can induce splenic iTregs and long-term tolerance to MBP with the potential to partially reverse the pathology of multiple sclerosis, particularly during the early stages of the disease., Funding: EMD Serono, Inc., a business of Merck KGaA.

Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (>4MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k(cat)(app)/K(M) probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K(M) and k(cat)(app) are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k(cat)(app), possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

The aim of this study was to investigate the mechanism by which tripeptidyl-peptidase II (TPP II) can specifically release tripeptides from the free N-terminus of an oligopeptide. The subtilisin-like N-terminal part of TPP II was modelled using subtilisin as template. Two glutamate residues (Glu-305 and Glu-331) appeared to be positioned so as to interact with the positively charged N-terminus of the substrate. In order to test this potential interaction, both residues were replaced by glutamine and lysine. The catalytic efficiency was reduced 400-fold for the E331Q variant and 20000-fold for the E331K variant, compared with the wild-type (wt). A substantial part of this reduction was due to decreased substrate affinity, since the K(M) for both mutants was at least two orders of magnitude greater than for the wt. This decrease was linked specifically to interaction with the free N-terminal amino group, based on inhibition studies. Glu-305 appears to be essential for enzymatic activity, but the extremely low activity of the E305Q variant prevented an investigation of the involvement of Glu-305 in substrate binding. The present work is, to our knowledge, the first report to investigate a mechanism for a tripeptidyl-peptidase activity through site-directed mutagenesis.

Tripeptidyl-peptidase II (TPP II) is one of the many proteases involved in the important process of intracellular proteolysis. The widespread distribution and broad substrate specificity suggest that TPP II is encoded by a "house-keeping gene". However, both TPP II protein and mRNA levels vary in different cells. To investigate whether these variations are due to regulation on a genetic level, the promoter of the TPP2 gene has previously been identified. The promoter contains two inverted CCAAT-boxes and an E-box. By means of reporter assays and electrophoretic mobility shift assays the promoter has now been further characterized. It could be concluded that USF-1 (upstream stimulatory factor-1) binds to the E-box in the promoter. The transcription factors NF-Y and USF-1 are present in protein-DNA complexes of different sizes. Mutation of the E-box had no effect, indicating that only binding of NF-Y to the two CCAAT-boxes was important for activation of transcription. However, this does not exclude the possibility that USF-1 can play an important role in transcription in other types of cells. Furthermore, the region upstream of the promoter was investigated due to its ability to inhibit transcription. Several silencer elements were identified and we also showed that Oct-1 binds to one of these elements. Thus, this investigation reveals that TPP II expression could be regulated through both positive and negative regulatory elements. Further studies are required to establish the involvement of different genetic elements, and how the interplay between different transcription factors will affect the transcriptional rate in vivo.

Unlabelled: Tripeptidyl peptidase II (TPPII) is a high molecular weight exopeptidase important in inactivating extracellular cholecystokinin (CCK). Our aims were to study the anatomical localization of TPPII and CCK mRNA in the Cynomolgus monkey brain as a basis for a possible functional anatomical connection between enzyme (TPPII) and substrate (CCK) and examine if indications of changes in substrate availability in the human brain might be reflected in changes of levels of TPPII mRNA., Methods: mRNA in situ hybridization on postmortem brain from patients having had a schizophrenia diagnosis as compared to controls and on monkey and rat brain slices., Results: overlapping distribution patterns of mRNAs for TPPII and CCK in rat and monkey. High amounts of TPPII mRNA are seen in the neocortex, especially in the frontal region and the hippocampus. TPPII mRNA is also present in the basal ganglia and cerebellum where CCK immunoreactivity and/or CCK B receptors have been found in earlier studies, suggesting presence of CCK-ergic afferents from other brain regions. Levels of mRNAs for CCK and TPPII show a positive correlation in postmortem human cerebral cortex Brodmann area (BA) 10. TPPII mRNA might be affected following schizophrenia., Discussion: overall TPPII and CCK mRNA show a similar distribution in rat and monkey brain, confirming and extending earlier studies in rodents. In addition, correlated levels of TPPII and CCK mRNA in human BA 10 corroborate a functional link between CCK and TPPII in the human brain.

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

Tripeptidyl-peptidase II is a high-molecular weight peptidase with a widespread distribution in eukaryotic cells. The enzyme sequentially removes tripeptides from a free N-terminus of longer peptides and also displays a low endopeptidase activity. A role for tripeptidyl-peptidase II in the formation of peptides for antigen presentation has recently become evident, and the enzyme also appears to be important for the degradation of some specific substrates, e.g. the neuropeptide cholecystokinin. However, it is likely that the main biological function of tripeptidyl-peptidase II is to participate in a general intracellular protein turnover. This peptidase may act on oligopeptides generated by the proteasome, or other endopeptidases, and the tripeptides formed would subsequently be good substrates for other exopeptidases. The fact that tripeptidyl-peptidase II activity is increased in sepsis-induced muscle wasting, a situation of enhanced protein turnover, corroborates this biological role.

OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during repeated administration of 400 mg OSI-7836/kg on days 1, 4, 7, and 10. The majority of OSI-7836 in DNA from Calu-6 and H460 tumors of mice dosed with 1600 mg/kg was located at internal nucleotide linkages, similar to dFdC and ara-C. In conclusion, cell cycling studies supported the hypothesis that OSI-7836 cytotoxicity is dependent upon DNA synthesis. A validated LC-MS/MS assay was developed that could quantify OSI-7836 in DNA from tissues. The assay was used to show that OSI-7836 was incorporated in internal linkages in tumor DNA in a manner that was dose-independent at the doses tested and did not appear to accumulate during repeated dosing. The results suggest that if DNA incorporation is a toxic event, the relationships between administered dose, DNA incorporation, and toxicity are complex.

The oligopeptidase tripeptidyl-peptidase II (TPP II) is up-regulated Burkitt's lymphoma (BL) cells that overexpress the c-myc proto-oncogene and is required for their growth and survival. Here we show that overexpression of TPP II induces accelerated growth and resistance to apoptosis in human embryonic kidney 293 cells. This correlates with the appearance of multiple chromosomal aberrations, numerical and structural centrosome abnormalities, and multipolar cell divisions. Similar mitotic aberrations were also observed in a panel of BL lines and were suppressed, in parallel with TPP II down-regulation, upon reversion of BL-like characteristics in EBV-immortalized B lymphocytes carrying a tetracycline-regulated c-myc. Functional TPP II knockdown by small interfering RNA expression in BL cells caused the appearance of giant polynucleated cells that failed to complete cell division. Collectively, these data point to a role of TPP II in the regulation of centrosome homeostasis and mitotic fidelity suggesting that this enzyme may be a critical player in the induction and/or maintenance of genetic instability in malignant cells.

Tripeptidyl-peptidase II (TPP II) is a ubiquitously expressed exopeptidase. The expression of this enzyme is increased, e.g. in some tumor cells, but the regulation of the expression of the gene has not been investigated previously. The gene encoding human TPP II (TPP2) is 82 kb and consists of 30 exons. An 8 kb NcoI fragment covering the 5'-flanking region of the TPP2 gene, including the initiation codon, was cloned into a luciferase-containing reporter vector. Human embryonic kidney cells (HEK-293 cells) and murine fibroblasts (NIH3T3 cells) were transiently transfected with the construct. Through sequential deletions and analysis of short PCR-fragments, the promoter could be localized to a 215 bp fragment upstream of the initiation codon. This region is GC-rich, lacks a TATA-box and contains two inverted CCAAT-boxes and a GC-box. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoter fragment. The 85 bp 5'-end of the promoter fragment is essential for transcriptional activation. Out of this a 44 bp fragment suffices to compete with binding of nuclear proteins to the 215 bp fragment. Supershift assays demonstrated that the CCAAT-binding factor (CBF; NF-Y) is involved in the formation of a complex with the 215 bp fragment. Although Sp1 binds to the promoter fragment in vitro, it was found to bind to the 3'-end of the 215 bp fragment which is not essential for transcription. The potential role of Sp1 in transcription of TPP2 therefore remains to be established.

OSI-7836 (4'-thio-araC, T-araC) is a nucleoside analogue that shows efficacy against solid tumor xenograft models. We examined how the triphosphates of OSI-7836 (T-araCTP), cytarabine (araCTP), and gemcitabine (dFdCTP) affected the initiation of new DNA strands by the pol alpha primase complex. Whereas dFdCTP very weakly inhibited primase, both T-araCTP and araCTP potently inhibited this enzyme. Primase polymerized T-araCTP and araCTP more readily than its natural substrate, CTP, and incorporation resulted in strong chain termination. dFdCTP, araCTP, and T-araCTP inhibited pol alpha competitively with respect to dCTP. When exogenously added primentemplates were used, pol alpha incorporated all three analogues into DNA, and incorporation caused either weak chain termination (dFdCTP), strong termination (araCTP), or extremely strong termination (T-araC). Furthermore, pol alpha polymerized T-araCTP only nine-fold less well than dCTP, whereas it polymerized araCTP and dFdCTP 24- and 83-fold less well, respectively. The presence of these three analogues in the template strand resulted in significant pausing by pol alpha, although the site and severity of pausing varied between the analogues. During the elongation of primase-synthesized primers, a reaction that is thought to mimic the normal sequence of events during the initiation of new DNA strands, pol alpha polymerized all three compounds. However, incorporation of araCTP and dFdCTP resulted in minimal chain termination, while incorporation of T-araCTP still caused extremely strong termination. The implications of these results with respect to how these compounds affect cells are discussed.

OSI-211 (liposomal lurtotecan), was evaluated using several different dose schedules (1mg/kg, d1-5, 1.75 mg/kg d1, 3, 5 and 6 mg/kg d1, 8) in severe combined immunodeficient (SCID) mouse models of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) with early treatment (ET, days 6-8) or late treatment (LT, days 15-19), examining early and advanced disease, respectively. Due to the aggressive nature of the Molt-4 model, the ET and LT were accelerated to day 3 or 4 and day 8 post-implant, respectively. For each model, 2 x 10(7) (KBM-3B) or 1 x 10(7) (Molt-4, HL-60 and CEM) leukemia cells were injected intravenously into the tail vein. Each control and test group consisted of eight animals. All three schedules (1mg/kg qd1-5, 1.75 mg/kg d1, 3, 5 and 6 mg/kg d1, 8) increased the life span of OSI-211 treated animals in each model, with a tendency toward improved efficacy with the 6 mg/kg d1, 8 schedule. As a result, the activity of the 6 mg/kg d1, 8 schedule is detailed for each model. ET significantly (P<0.005) increased survival in the KBM-3B model with 86% long-term survivors (LTS). Using PRC analysis, human beta-globin gene sequences in one or several tissues were amplified in all but 3 LTS, suggesting minimal residual disease in 26 of the 29 LTS. LT also significantly (P<0.005) improved average life span in the KBM-3B model, with an average ILS=196+/-11% and one LTS. Treatment of HL-60 leukemia animals significantly (P<0.005) increased life span, with an ILS=213+/-9% and two LTS for ET, and with an ILS=219+/-4% and no LTS for LT. Treatment of Molt-4 animals, the most aggressive leukemia model tested, significantly (P<0.005) increased life span, with an average ILS=181+/-3% and no LTS for ET and an average ILS=172+/-1% with no LTS for LT. In the CEM model, ET resulted in a significantly (P<0.005) improved ILS=244+/-24% with one LTS. In comparison to OSI-211, treatment with DaunoXome, the liposomal formulation of daunorubicin, a drug with clinical efficacy in AML and ALL, had no effect on survival in the KBM-3B, nor Molt-4 A4 leukemia models when administered at its maximum or near maximum tolerated doses of 3mg/kg d1, 8. These data demonstrate that OSI-211 has potent antileukemia activity in preclinical SCID mouse AML and ALL leukemia models, supporting the clinical investigation of OSI-211 for hematological malignancies.

Tripeptidyl-peptidase II (TPP II) is a 138-kDa subtilisin-like serine peptidase forming high molecular mass oligomers of >1000 kDa. The enzyme participates in general protein turnover and apoptotic pathways, and also has specific substrates such as neuropeptides. Here we report the site-directed mutagenesis of amino acids predicted to be involved in catalysis. The amino acids forming the putative catalytic triad (Asp-44, His-264, Ser-449) as well as the conserved Asn-362, potentially stabilizing the transition state, were replaced by alanine and the mutated cDNAs were transfected into human embryonic kidney (HEK) 293 cells. In clones stably expressing the mutant proteins, TPP II activity did not exceed the endogenous activity, thus confirming the essential role of the above amino acids in catalysis. Mutant and wild-type TPP II subunits co-eluted from a gel filtration column, suggesting that the subunits associate and that the native subunit conformation was retained in the mutants. Interestingly, the S449A and a H264A mutant enzyme affected the quaternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of >10,000 kDa.