Thrina Loennechen

Interethnic difference in thiopurine methyltransferase activity

A number of metabolic pathways are subject to both genetic polymorphism and interethnic differences. A catabolic pathway of 6‐mercaptopurine, red blood cell (RBC) thiopurine methyltransferase (TPMT) activity showed genetic polymorphism in Caucasians, but variation according to ethnicity has not been studied. We investigated if red blood cell thiopurine methyltransferase was subject to interethnic variation in a Saami (Lappish; n = 36) and a Caucasian population (n = 50). The Saami population sample had 29% higher thiopurine methyltransferase activity, 17.0 ±3.3 U/ml red blood cell compared with the Caucasian population sample, 13.1 ± 2.9 U/ml red blood cell (p < 0.001). Probit plots and frequency distribution histograms supported bimodality consistent with genetic polymorphism in both study populations. Differences in chronic diseases, drug consumption, age, or gender could not explain the interethnic difference in red blood cell thiopurine methyltransferase activity. The higher red blood cell thiopurine methyltransferase activity in the Saami population group indicates that these subjects may require higher dosages of thiopurine drugs than Caucasians.

Detection of one single mutation predicts thiopurine S-methyltransferase activity in a population of Saami in northern Norway

Thiopurine S‐methyltransferase (TPMT) activity exhibits genetic polymorphism. The purpose of this investigation was to identify TPMT mutant alleles in the Saami population as a basis of developing genotyping tests for prediction of TPMT activity. The most predominant allele in Saamis (n = 194) was the TPMT*3C allele (A719G mutation) representing 92% of the mutant alleles, with an estimated allelic frequency of 3.3%. The most frequent allele in Caucasians (n = 66) living in the same geographic area was the TPMT*3A (A719G and G460A mutations) representing 91% of the mutant alleles, with an estimated allelic frequency of 3.4%. A test for one mutation, A719G, may prospectively identify more than 90% of the Saami individuals who require reduction in thiopurine dose to avoid hematopoietic toxicity. In a Norwegian population, comprising both the major Caucasian population and a minor Saami population, the same genotyping tests (eg, tests for the A719G and G460A mutations) may be used.

The interaction of 6-mercaptopurine (6-MP) and methotrexate (MTX).

The antimetabolites 6-mercaptopurine (6-MP) and methotrexate (MTX) are the cornerstones in the maintenance treatment of childrens acute lymphoblastic leukemia (ALL). The biochemical mechanisms underlying the increased therapeutic efficacy of the combination of these drugs have not yet been elucidated. However, both drugs interact with important pathways. such as purine de novo synthesis (PDNS), purine salvage, and methylation reactions. A review of the mechanistic aspects of the interactions between 6-MP and MTX is given.

Synergistic increase in c-fos expression by simultaneous activation of the ras/raf/map kinase- and protein kinase A signaling pathways is mediated by the c-fos AP-1 and SRE sites.

Expression of the c-fos proto-oncogene is induced by numerous stimuli some of which are transmitted through the Ras/Raf/MAP kinase or the cAMP-dependent protein kinase (PKA) pathways. The effect of cell-specific interactions between these pathways on c-fos expression was investigated by exposing quiescent NIH3T3 cells to serum, forskolin, or a combination. Co-stimulation with serum and forskolin resulted in a more than additive increase in c-fos transcription. Synergistic increase in c-fos promoter activity was also observed in transient transfection studies after co-stimulation with serum plus forskolin or co-transfection with c-Raf and PKA expression plasmids. Analysis of the cAMP signaling pathway revealed that the synergy was neither due to an increase in PKA activity nor to Ser-133 phosphorylation/activation of CREB. The activation status of the MAP kinases ERK1 and ERK2 in co-treated cells was comparable to that in serum-treated cells. Co-stimulation with forskolin did not alter the phosphorylation state of Elk-1 compared to serum-induced phosphorylation of Elk-1. Deletion of c-fos promoter elements previously shown to be important for regulation of c-fos expression in response to mitogens indicates a role for SRE and FAP-1 elements.

Increased Concentrations of Methylated 6-Mercaptopurine Metabolites and 6-Thioguanine Nucleotides in Human Leukemic Cells In Vitro by Methotrexate

The effect of methotrexate (MTX) on 6-mercaptopurine (6-MP) metabolism was studied in four human leukemic cell lines in vitro. CCRF-CEM, WI-L2, TBJ, and HL-60 all expressed thiopurine methyltransferase (TPMT) activity. The cells were grown in horse serum-supplemented RPMI 1640 medium to which was added 4 microM of 6-MP or 4 microM of 6-MP and 20 nM of MTX. The presence of MTX resulted in a 2.1-, 1.7-, 2.4- and 8-fold increase in the concentrations of methylmercaptopurine ribonucleotides (MMPRP) in CEM, WI-L2, TBJ, and HL-60 cells, respectively (P < 0.0008). The concentrations of 6-thioguanine nucleotides (6 TGN) increased 1.9-, 1.4-, 2.4- and 1.9-fold in the same cell lines (P < 0.02). The four cell lines differed with respect to the effect of MTX on the consumption of 6-MP from the medium; CEM consumed more 6-MP and WI-L2 less 6-MP from media containing MTX than from media containing 6-MP only (P = 0.005 and 0.02, respectively). MTX did not affect the consumption of 6-MP by TBJ cells (P = 0.17). Media in which HL-60 cells had been grown did not contain detectable amounts of 6-MP at the end of the experiment. The simultaneous increase in methylated 6-MP metabolites and 6-TGN represents a possible explanation for the synergism of MTX and 6-MP; however, the clinical importance of increased MMPRP remains to be elucidated.

Adherence to medication guideline criteria in cancer pain management

The medication-assessment tool for cancer pain management (MAT-CP) is a novel tool for measuring the quality of drug use in chronic pain management in relation to guideline standards. MAT-CP has recently been revised and validated for use in the U.K. clinical setting. This article presents a measure of the adherence of current practice to specific cancer pain guideline criteria in two palliative care settings. Adult patients with malignant disease experiencing pain and/or receiving analgesics were identified by clinical pharmacists at two hospitals and five hospices in Scotland, United Kingdom. The MAT-CP was applied to data extracted from case notes. Results were quantified in terms of applicability and adherence to guideline criteria and the presence of insufficient data. MAT-CP was applied to 192 cancer patients experiencing pain; 103 (54%) were males and the mean (standard deviation) age was 68.5 (13.0) years. Overall guideline adherence was 75% (confidence interval [CI]: 74%, 77%; n=3460 applicable criteria). Low adherence (<50%) was seen for nine criteria, whereas 21 criteria were considered high-adherence criteria (>75%). Overall adherences for 56 (29%) hospitalized patients and 136 (71%) hospice patients were 65% (CI: 62%, 68%) and 79% (CI: 78%, 81%), respectively. Although good overall guideline adherence was found, there were gaps in both the hospice and hospital palliative care settings in the implementation of certain treatment recommendations, particularly in relation to pain assessment. The application of the tool has highlighted issues for feedback to health care providers and for further study.

Analysis of methylated 6-mercaptopurine metabolites in human red blood cells: comparison of two methods.

Blood samples from 34 recipients of kidney transplants who were on multidrug therapy including azathioprine were analyzed using two methods in parallel for red blood cell (RBC) concentrations of methylated 6-mercaptopurine (6-MP) metabolites. Chemical hydrolysis with high-performance liquid chromatography (HPLC) showed values ranging from 0 to 20,259 pmol/8 x 10(8) RBCs, compared with enzymatic hydrolysis with HPLC that resulted in values ranging from 16 to 22,252 pmol/100 microl packed RBC. Results of the two methods were highly correlated; the coefficient of correlation (r) was equal to 0.93 (95% confidence interval [CI] = 0.87-0.97 [y = 1.12x + 187]). Within series imprecision was 3.1% compared with 6.3%, and between-run imprecision was 10.3% compared with 20.7%, for the enzymatic and chemical methods, respectively. The enzymatic method was found to be more specific and to save time and labor, but with the chemical method methylated metabolites and 6-thioguanine nucleotides (6-TGN), the main active metabolites of azathioprine and 6-MP, can be measured in the same run. The results indicate that methylated 6-MP metabolites mainly exist as ribonucleotides in RBCs.

Dysregulation of Matrix Metalloproteinases and their Tissue Inhibitors by S100A4

The S100A4 protein as well as the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are associated with diseases such as arthritis and cancer. This mini review focuses on in vitro and in vivo studies indicating S100A4 involvement in regulation of MMPs and TIMPs, and the biological and pathobiological consequences of this regulation.

Effect of adenosine analogues on the expression of matrix metalloproteinases and their inhibitors from human dermal fibroblasts

The effect of the cytostatic and antiviral adenosine analogues 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on human skin fibroblasts was studied. Variables examined were cell morphology, viability, DNA fragmentation, expression of matrix metalloproteinases (MMPs) and matrix metalloproteinase inhibitors (TIMPs). None of these variables were changed when cells were exposed to c3Ari concentrations ranging from 10(-5) to 10(-3) M or 10(-5) M c3Ado. However, large changes in cell morphology, viability and expression of MMPs and MMP inhibitors occurred when fibroblasts were treated with 10(-4) or 10(-3) M c3Ado. Cells rounded up, shrank in volume, some detached and viability was lost without any detectable fragmentation of DNA. These changes in morphology and viability were associated with a differentiated expression of MMPs and MMP inhibitors. A large increase in collagenase activity occurred, and depending on the concentration of the adenosine analogue and the length of treatment, this change in activity could be shown to be due to one or a combination of the following factors: an increased synthesis of the collagenase protein, a decreased production of TIMP-1 or an increased activity of the collagenase superactivator, stromelysin. In contrast to this, treatment with c3Ado resulted in a decreased gelatinase activity, which in part could be attributed to an increased production of an inhibitor that seemed to affect gelatinase but not collagenase. The cellular changes induced by c3Ado seemed to reflect some of the alteration in the metabolic machinery that appears during a drug-induced or programmed/controlled death of a dermal cell. The different effects exerted by these two adenosine analogues on dermal fibroblasts can at least in part explain why c3Ado have previously been shown to be more toxic than c3Ari in animal models.

Collagen I regulates matrix metalloproteinase-2 activation in osteosarcoma cells independent of S100A4.

This work investigates the effect of cell–collagen I interactions on the synthesis and activation of MMP‐2, as well as synthesis of MT1‐MMP and TIMP‐1, by using an in vitro model with 3D fibrillar and 2D monomeric collagen. In order to reveal whether the metastasis‐associated protein S100A4 can influence the cell’s response to the two forms of collagen, osteosarcoma cell lines with high and low endogenous levels of S100A4 were used. Attachment of osteosarcoma cells to 3D fibrillar and 2D monomeric collagen resulted in opposite effects on MMP‐2 activation. Attachment to 3D fibrillar collagen decreased activation of proMMP‐2, with a corresponding reduction in MT1‐MMP. By contrast, attachment to monomeric collagen increased the amount of fully active MMP‐2. This was caused by a reduction in TIMP‐1 levels when cells were attached to monomeric 2D collagen. The effect of collagen on proMMP‐2 activation was independent of endogenous S100A4 levels, whereas synthesis of TIMP‐1 was dependent on S100A4. When cells were attached to monomeric collagen, cells with a high level of S100A4 showed a greater reduction in the synthesis of TIMP‐1 than did those with a low level of S100A4. Taken together, this study shows that synthesis and activation of MMP‐2 is affected by interactions between osteosarcoma cells and collagen I in both fibrillar and monomeric form.