Berta Otová

Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells

L-Asparaginase is a standard component in chemotherapy of childhood acute lymphoblastic leukaemia (ALL). Leukaemic cells carrying TEL/AML1 fusion gene are more sensitive to treatment with L-asparaginase compared to other subtypes of ALL. We demonstrate in vitro the prolonged growth suppression of TEL/AML1[+] cells compared to TEL/AML1[−] leukaemic cells after L-asparaginase treatment simulating treatment protocol. Cell cycle analysis revealed TEL/AML1[+] cells to accumulate in G1/G0 phase (81–98%) compared to TEL/AML1[−] cells (47–60%). Quantitative analysis of asparagine synthetase (AsnS) expression showed the ability of TEL/AML1[+] cells to increase AsnS mRNA levels after L-asparaginase treatment to the same extent as TEL/AML1[−] leukaemic and nonleukaemic lymphoid cells. We hypothesise that TEL/AML1[+] cells are unable to progress into the S phase of cell cycle under nutrition stress caused by L-asparaginase, despite the ability of AsnS upregulation. Significantly higher expression of AsnS was found in untreated leukaemic cells from children with TEL/AML1[+] ALL (n=20) in comparison with the group of age-matched children with ALL bearing no known fusion gene (n=25; P=0.0043). Interestingly, none of the TEL/AML1[+] patients with high AsnS level relapsed, whereas 10/15 patients with AsnS below median relapsed (P=0.00028). Therefore, high AsnS levels in TEL/AML1[+] patients correlate with better prognosis, possibly reflecting the stretched metabolic demand of the lymphoblast.

Slower molecular response to treatment predicts poor outcome in patients with TEL/AML1 positive acute lymphoblastic leukemia: Prospective real-time quantitative reverse transcriptase-polymerase chain reaction study

The translocation t(12;21)(p13;q22), which produces the TEL/AML1 fusion gene, is the most frequent chromosomal abnormality in patients with childhood acute lymphoblastic leukemia (ALL) and generally is associated with a favorable prognosis. Furthermore, real‐time quantitative‐polymerase chain reaction (RQ‐PCR)‐based detection of TEL/AML1 represents an accurate technique for the reproducible assessment of minimal residual disease (MRD).

Tenofovir Diphosphate Is a Poor Substrate and a Weak Inhibitor of Rat DNA Polymerases α, δ, and ε*

ABSTRACT Tenofovir diphosphate (PMPApp) is a weak inhibitor of DNA polymerases (pol) α, δ, and ε*, with values for the Ki for PMPApp (PMPAppKi) relative to the Km for dATP (dATPKm) of 10.2, 10.2, and 15.2, respectively. Its incorporation into DNA was about 1,000-fold less efficient than that of dATP, with PMPAppKm values 350-, 2,155-, and 187-fold higher than dATPKm values for pol α, δ, and ε*, respectively.

9-[2-(phosphonomethoxy)ethyl]adenine diphosphate (PMEApp) as a substrate toward replicative DNA polymerases α, δ, ε, and ε∗

Abstract The diphosphoryl derivative of the acyclic nucleotide phosphonate analog 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), found previously to weakly inhibit DNA pol δ/proliferating cell nuclear antigen, was studied as a substrate for pol α, δ, e, and e∗. A comparison of the V max and K m for this derivative (PMEApp) and dATP demonstrated that the relative efficiency of the incorporation of this analog into the DNA chain is decreasing in the following order: pol δ ≃ pol e ≃ pol e∗ > pol α. Under the reaction conditions, this incorporation amounted to 4.4 to 0.7% of dAMP molecules. Similar K m values for PMEApp and dATP in pol e and pol e∗ catalyzed reactions revealed that proteolysis of the enzyme probably does not affect the dNTP binding site. The DNA polymerases tested were inhibited by the reaction product (PMEA terminated DNA chain) with similar K i / K m ratios (pol α 0.2; pol δ, 0.1; pol e 0.05; and pol e∗, 0.06). The associated 3′-5′-exonuclease activity of pol δ, e, and e∗ was able to excise PMEA from the 3′-OH end of DNA with a rate one order of magnitude lower than that of the dAMP residue.

Suppression of rat adjuvant arthritis by some acyclic nucleotide analogs

The antiarthritic potential of two different acyclic nucleotide analogs, i.e. 9-(2-phosphonomethoxyethyl)adenine (PMEA), its bis(pivaloyloxymethyl)ester (Bis-POM-PMEA), and 1-(S)-(3-hydroxy-2-phosphonomethoxyethyl) cytosine (HPMPC) was investigated in the rat model of mycobacterial adjuvant-induced arthritis. With dependence on the dose, timing and route of administration, as well as on the genetic constitution of the arthritis-prone animals, PMEA was able to delay the onset, and substantially reduce or nearly completely inhibit the development of arthritic paw swelling. HPMPC was less active in this model. As compared with PMEA, its prodrug, Bis-POM-PMEA, expressed much more pronounced beneficial effects after both oral and i.p. administration.

Second-generation taxanes effectively suppress subcutaneous rat lymphoma: role of disposition, transport, metabolism, in vitro potency and expression of angiogenesis genes

SummaryThe study investigated possible mechanisms by which second-generation taxanes, established as significantly more effective than paclitaxel in vitro, suppress a rat lymphoma model in vivo. The studied mechanisms included taxane pharmacokinetics, expression of genes dominating their metabolism (Cyp3a1/2) and transport (Abcb1) and genes controlling tumour angiogenesis (growth factors and receptors). SB-T-1214, SB-T-12854 and IDN5109 suppressed rat lymphoma more effectively than paclitaxel, SB-T-12851, SB-T-12852, SB-T-12853 or IDN5390 as well as P388D1 leukaemia cells in vitro. The greater anti-lymphoma effects of SB-T-1214 in rats corresponded to a higher bioavailability than with SB-T-12854, and lower systemic toxicity of SB-T-1214 for rats reflected its lower cytotoxicity for P388D1 cells in vitro. Suppression of Abcb1 and CYP3a1 expression by SB-T-1214 and IDN5109 could partly explain their anti-lymphoma effects, but not that of SB-T-12854. Growth factors genes Egf, Fgf, Pdgf, and Vegf associated with tumour angiogenesis had significantly lower expression following treatment with anti-lymphoma effective IDN5109 and their receptors were unaffected, whereas inefficient IDN5390 increased expression of the most important Vegf. The effective SB-T-12854 inhibited Egf, Egfr, Fgfr and Pdgfr expression, while the ineffective SB-T-12851, SB-T-12852 and SB-T-12853 inhibited only Egf or Egfr expression. Vegfr expression was inhibited significantly by SB-T-12851 and SB-T-12854, but effect of SB-T-12851 was compromised by induced Vegf expression. The very effective SB-T-1214 decreased the expression of Vegf, Egf and all receptors most prominently indicating the possible supporting role of these genes in anti-lymphoma effects. In conclusion, SB-T-1214, SB-T-12854 and IDN5109 are good candidates for further study.

Reply to ‘Upregulation of asparagine synthetase and cell cycle arrest in t(12;21) positive ALL’ by Stams et al

Reply to ‘Upregulation of asparagine synthetase and cell cycle arrest in t(12;21) positive ALL’ by Stams et al

Reply to Stams et al

84: 3473–3482. 4 Stams WA, den Boer ML, Beverloo HB, Meijerink JP, Stigter RL, van Wering ER et al. Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21)+ pediatric ALL. Blood 2003; 101: 2743–2747. 5 Stams WA, den Boer ML, Beverloo HB, Meijerink JP, Van Wering ER, Janka-Schaub GE et al. Expression levels of TEL, AML1 and the fusion products TEL-AML1 and AML1-TEL versus drug sensitivity and clinical outcome in t(12;21) positive pediatric ALL. submitted. 6 Hubner S, Cazzaniga G, Flohr T, van der Velden VH, Konrad M, Potschger U et al. High incidence and unique features of antigen receptor gene rearrangements in TEL-AML1-positive leukemias. Leukemia 2004; 18: 84–91.

Abstract B9: Role of disposition, transport, in vitro efficiency and expression of relevant genes in the effects of novel taxanes on subcutaneous lymphoma in Sprague‐Dawley/Cub rats

Novel taxoids SB‐T‐1214, SB‐T‐12851, SB‐T‐12852, SB‐T‐12853, SB‐T‐12854 and IDN‐5109 proved to be more effective than paclitaxel in vitro in MDA‐MB‐435 cell line, but especially in NCI/ADR‐RES cell line, highly expressing ABCB1. Thus, their effects in vivo are of considerable interest, because they could overcome drug‐resistance due to high ABCB1 and CYP3A expressions which may limit effects of paclitaxel. Purposes of the study: To investigate if some of the novel taxanes synthesized at Stony Brook exert significant effects on the genetically well defined lymphoma in Sprague‐Dawley/Cub rats, for which paclitaxel is ineffective. Experimental procedures: The rats were kept in standard laboratory conditions and the drugs were administered i.p. under conditions approved by Ethical Committee, Charles University Prague. The intravital area of subcutaneous lymphoma was measured by palpation and their weight during post‐mortem examination. Drug blood levels were determined by HPLC. Quantitative expressions of mRNAs of Abcb1, Cyp3a, Egf, Fgf, Pdgf and Vegf and their receptors Egfr, Fgfr, Pdgfr and Vegfr were made by real‐time PCR system using reaction mixtures of Applied Biosystems. Effects were also compared with in vitro efficiency of the drugs in P388D1 lymphoma line. Summary of new and unpublished data: IDN‐5109, SB‐T‐1214 and SB‐T‐12854 were the most effective drugs against the subcutaneous lymphoma. The higher effects of SB‐T‐1214 than SB‐T‐12854 are ascribed to 3.3‐higher blood levels, as well as to the fact that systemic toxicity of SB‐T‐1214 is significantly lower than that of SB‐T‐12854. Expression of Abcb1 and Cyp3a1 in lymphomas after treatment with the drugs does not correspond to their anti‐lymphoma effects. However, the anti‐lymphoma activities of IDN‐5109, SB‐T‐1214 and SB‐T‐12854 corresponded to their effects on growth factors and their receptors, especially Vegf and Vegfr. The result is consistent with the reported effect of paclitaxel on growth factors and tumor angiogenesis, and these effects could be an important factor for their activities in addition to the proved effects on mitosis and apoptosis. Higher in vitro activities of the novel taxanes than paclitaxel in P388D1 cell line correspond to their higher efficacies on the subcutaneous lymphoma in vivo as compared with paclitaxel. Conclusions: IDN‐5109, already proved effective against various tumors and presently in Phase II clinical studies, as well as novel taxanes SB‐T‐1214 and SB‐T‐12854, are found to be effective against non‐Hodgkin type rat lymphoma. These taxanes could be efficacious against paclitaxel‐resistant tumors, due to the fact that they are not subjected to drug‐resistance caused by high ABCB1 expression and are potentially effective against tumor angiogenesis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B9.