Increased CK Activity Research Articles

Methodology-related pseudohyperkalemia associated with marked muscle enzyme leakage in a dog.

As hyperkalemia may be life-threatening, it is critical to recognize artifactually increased potassium concentrations. Pseudohyperkalemia may occur in myopathies when using the VetScan2 analyzer (VS2), but the degree of pseudohyperkalemia and relationships relative to creatine kinase activity (CK) are unknown. We aimed to determine what degree of muscle enzyme leakage, as reflected by increased serum CK activity, results in cases with falsely elevated potassium concentrations when measured by the VS2. We also sought to establish if a linear relationship exists between potassium concentrations measured by the VS2 and CK activity. Serum samples from dogs with increased CK activity and with CK activity within the reference interval and without clinically relevant biochemical alterations were used to create diluted samples having various CK activities. Potassium concentrations and CK activities were determined on VS2 and Cobas c501 (Cobas) analyzers. Wilcoxon signed rank, Bland-Altman, and Passing-Bablok analyses were used to compare potassium concentrations generated by the VS2 and Cobas analyzers. Least squares regression analysis was performed to evaluate if a linear relationship exists between VS2 potassium concentrations and Cobas CK activities. Potassium concentrations from the VS2 were higher (median and standard deviation (SD) = 5.2 +/- 0.46 mmol/L) than those from the Cobas analyzer (4.4 +/- 0.35 mmol/L; P < 0.000), and a positive mean bias was found (mean difference = 0.774 mmol/L; 95% Confidence Interval (CI) = 0.706-0.842; limits of agreement = 0.21-1.34). Passing-Bablok regression showed a positive proportional bias for potassium concentrations on the VS2 compared with paired Cobas results (Slope = 1.328; 95% CI = 1.100-1.500) but did not reveal systematic bias (Intercept = -0.714; 95% CI = -1.46-0.265). Least squares regression analysis showed a poor non-significant relationship (R2 = 0.19) between potassium measured by the VS2 and CK measured by the Cobas analyzer. A defined threshold value of CK activity at which potassium concentration begins to falsely increase when measured on the VS2 was not established as data widely varied. A poor non-significant relationship between potassium concentrations and CK activities did not allow prediction of the threshold at which falsely increased potassium concentrations would be expected on the VS2.

SiRNA knockdown of mitochondrial thymidine kinase 2 (TK2) sensitizes human tumor cells to gemcitabine.

Nucleoside metabolism enzymes are determinants of chemotherapeutic drug activity. The nucleoside salvage enzyme deoxycytidine kinase (dCK) activates gemcitabine (2', 2'-difluoro-2'-deoxycytidine) and is negatively regulated by deoxycytidine triphosphate (dCTP). Reduction of dCTP in tumor cells could, therefore, enhance gemcitabine activity. Mitochondrial thymidine kinase 2 (TK2) phosphorylates deoxycytidine to generate dCTP. We hypothesized that: (1) TK2 modulates human tumor cell sensitivity to gemcitabine, and (2) antisense knockdown of TK2 would decrease dCTP and increase dCK activity and gemcitabine activation. siRNA downregulation of TK2 sensitized MCF7 and HeLa cells (high and moderate TK2) but not A549 cells (low TK2) to gemcitabine. Combined treatment with TK2 siRNA and gemcitabine increased dCK. We also hypothesized that TK2 siRNA-induced drug sensitization results in mitochondrial damage that enhances gemcitabine effectiveness. TK2 siRNA and gemcitabine decreased mitochondrial redox status, DNA content, and activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 function from that of other dTMP-producing enzymes [cytosolic TK1 and thymidylate synthase (TS)]. siRNA knockdown of TK1 and/or TS did not sensitize cancer cells to gemcitabine indicating that, among the 3 enzymes, only TK2 is a candidate therapeutic target for combination with gemcitabine.

THU0478 MRI Quantitative Analysis of Fatty Infiltration of Paravertebral Muscles in Patients with Primary Camptocormia

BackgroundCamptocormia also referred to as “bent spine syndrome” is characterized by an abnormal trunk posture with marked flexion of the thoraco-lumbar spine. These clinical signs are most prominent on standing...

Effects of Shenmai-injectio postconditioning on myocardial ischemia-reperfusion injury in rats

Objective To investigate the effects of postconditioning with Shenmai-injectio on myocardial ischemia-reperfusion (I/R) injury in rats.Methods Thirty-six healthy male SD rats aged 10-12 weeks weighing 240-260 g were randomly divided into 3 groups ( n =12 each):sham operation group (group S) ; myocardial I/R group and Shenmai-injectio postconditioning group (group SPO).Myocardial I/R was produced by ligation of the left anterior descending branch of coronary artery for 30 min followed by 120 min reperfusion in groups I/R and SPO.In group SPO Shenmai-injectio 9 ml/kg was injected iv at the end of 30 min ischemia.Blood samples were collected from abdominal aorta at the end of 120 min reperfusion for determination of serum CK activity and cTnI concentration.The animals were then sacrificed.Myocardial specimens were obtained for microscopic examination,detection of apoptosis and determination of myocardial Bcl-2 and Bax protein expression ( by immuno-histochemis-try).Results Myocardial I/R significantly increased serum CK activity,cTnI concentration,apoptotic index (percentage of apoptotic cells) and Bax protein expression and decreased Bcl-2 protein expression in group I/R as compared with group S.Shenmai-injectio postconditioning significantly attenuated I/R-induced above changes and ameliorated histo-pathological damage in group SPO as compared with group I/R.Conclusion Shenmai-injectiopostconditioning can reduce myocardial I/R injury by up-regulating Bcl-2 expression and down-regulating Bax expression,leading eventually to reduction in apoptosis. Key words: Myocardial reperfusion injury; Shenmai injection; Postconditioning

Staurosporine increases toxicity of gemcitabine in non-small cell lung cancer cells: role of protein kinase C, deoxycytidine kinase and ribonucleotide reductase

Gemcitabine, a deoxycytidine analog, active against non-small cell lung cancer, is phosphorylated by deoxycytidine kinase (dCK) to active nucleotides. Earlier, we found increased sensitivity to gemcitabine in P-glycoprotein (SW-2R160) and multidrug resistance-associated protein (SW-2R120), overexpressing variants of the human SW1573 non-small cell lung cancer cells. This was related to increased dCK activity. As protein kinase C (PKC) is higher in 2R120 and 2R160 cells and may control the dCK activity, we investigated whether gemcitabine sensitivity was affected by the protein kinase C inhibitor, staurosporine, which also modulates the cell cycle. Ten nmol/l staurosporine enhanced the sensitivity of SW1573, 2R120 and 2R160 cells 10-fold, 50-fold and 270-fold, respectively. Staurosporine increased dCK activity about two-fold and the activity of thymidine kinase 2, which may also activate gemcitabine. Staurosporine also directly increased dCK in cell free extracts. Staurosporine decreased expression of the free transcription factor E2F and of ribonucleotide reductase (RNR), a target for gemcitabine inhibition. In conclusion, staurosporine may potentiate gemcitabine by increasing dCK and decreasing E2F and RNR, which will lead to a more pronounced RNR inhibition.

“Necklace” fibers as a late clue to the interpretation of the forgotten “trilaminar” fibers

In a recent article by Bevilacqua et al. [1] on ‘‘necklace fibers, a new histological marker of late-onset MTM1related centronuclear myopathy’’, impressive light micrographs of such fibers are presented which closely resemble those previously described as ‘‘trilaminar’’ fibers [2–4], as far as the different techniques applied at the earlier and present times allow identification. Both the clinical and the structural changes appear rather similar within a wide spectrum of individual variations. Ringel et al. [2] in 1978 reported on a sporadic myopathy with pronounced rigidity, lack of spontaneous movements, and increased CK values. Electromyography appeared to be normal. The muscle biopsy at the age of 7 weeks showed numerous fibers with three concentric zones leading to the designation as trilaminar fibers. The innermost zone showed accumulations of mitochondria, glycogen, and electron dense material with only few remnants of myofibrils. The intermediate zone consisted of myofibrils showing Z-band streaming. The outer zone resembled a sarcoplasmic mass. Histochemistry revealed extrajunctional acetylcholine receptor (AChR) between the intermediate and outer zone. Increased muscle tone was attributed to a disturbed neural influence, while increased CK activity was interpreted as being more characteristic for a primary muscle lesion. The female case of Schroder and Schonberger published in 1981 [3, 4] was clearly a familial one, deceasing at the age of 18 months from bronchopneumonia. The older brother was born with bilateral club feet dying at the age of 3 months due to aspiration pneumonia. A younger brother showed delayed motor milestones with tremor since birth. He died at the age of 9 months. One sister and the parents appeared to be unaffected. Therefore, autosomal recessive inheritance was assumed. Muscle biopsy showed at the light microscopic level in semithin sections many muscle fibers with an outer light zone, an intermediate darker ring, and a relatively light center (Fig. 1a–d). At the electron microscopic level (Fig. 2a, b), there were glycogen granules, mitochondria, components of the T-system and microtubules in the outer zone. The intermediate ring showed an increased density of myofibrillar structures, while the center was rather disorganized. The nuclei were located in the peripheral, not in the central zone similar to those in the recent biopsies. The trilaminar fibers were smaller than the normal ones but not severely atrophic. To me, there appears to be no essential difference between ‘‘necklace’’ and ‘‘trilaminar’’ fibers, although the published electron micrographs of necklace fibers show less impressive or representative changes than the light micrographs and the electron micrographs previously published of the trilaminar fibers. The anti-desmin and antiaB-crystalline labelling of the intermediate ring in necklace fibers corresponds quite closely to the electron dense intermediate zone of trilaminar fibers, which I would have liked to call ‘‘cockade’’ (in French ‘‘cocarde’’, in German: Kokarden-) fibers if there would not have been the national emblematic implication. ‘‘Trilaminar’’ or ‘‘necklace’’ fibers are obviously very rare because in 40 years (between 1965 and 2005) looking at more than 12,000 muscle biopsies there was not a single other one with similar changes, including myotonic dystrophy. Nevertheless, it is highly appreciated that the changes have now been attributed to a distinct molecular genetically defined myopathy, which is due to mutations in the phosphoinositide phosphatase myotubularin 1 protein (MTM1) gene [1]. J. M. Schroder (&) Department of Neuropathology, Aachen University Hospital, RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany e-mail: jmschroder@ukaachen.de

Synergistic interaction between rituximab and 2-chlorodeoxyadenosine (2-CdA) against Waldenstrom's Macroglobulinemia preclinical model

2529 Background: Waldenstrom's Macroglobulinemia (WM) is an incurable malignant disorder. Rituximab represents a new therapeutic modality for B-cell tumors and has modest activity in WM. Among cytotoxic agents, 2-CdA has significant activity. In this study, Rituximab and 2-CdA combination was used in pre-clinical SCID mouse xenograft model of human WM (WSU-WM-SCID) (Blood 81:3034, 93). Methods: Subcutaneous (sc) tumor-bearing ICR-SCID mice were randomly assigned to one of four groups (8 animals each): Group 1 (control); Group 2 received Rituximab intravenously (iv) via tail vein daily x 5 days at a dose of 150 mg/kgm/day; Group 3 received 2-CdA injections sc daily x 5 days at MTD of 30 mg/kg/d; Group 4 received a combination of Rituximab and 2-CdA, as in groups 2 and 3, concurrently. Two treatment cycles were given with one week rest in between. Subsequent experiments focused on biological studies and involved removing tumors following therapy for flow cytometry and deoxycytidine kinase (dCK) activity. Results: Both agents showed modest activity in this model [Rituximab T/C = 30%, T-C = 13 days, Log10kill 1.9; 2CdA T/C = 36.9%, T-C = 14 days, Log10kill 1.33] but tumors progressed within 1 week. Residual tumors following Rituximab therapy preserved original phenotype except were CD20(-) due to Rituximab binding as confirmed by anti-Rituximab antibody. When used in combination, 8/8 animals were cured (no tumor development for 120 days). Treatment was well tolerated. Rituximab therapy increased dCK activity by 170%, 210%, and 258% after 1, 3, and 5 injections respectively. dCK is key to the phosphorylation of 2-CdA to its active metabolite 2-CdATP. Conclusions: there is synergistic interaction between Rituximab and 2-CdA leading to cure in this WM model. Enhancing dCK activity is one mechanism of interaction between the two agents. The WSU-WM-SCID is a model of resistant WM where no cures were achieved using standard agents until now. Clinical use of the combination against WM is recommended. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration International Waldenstrom's Macroglobulinemia Foundation

Selective activation of deoxycytidine kinase by thymidine-5′-thiosulphate and release by deoxycytidine in human lymphocytes

Deoxycytidine kinase (dCK) catalyses the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity [Biochem Pharmacol 56 (1998) 1175], improving the efficacy of the cytostatic therapy. Here, we measured the effect of the 5′-thiosulphate (5′-TS) derivatives of four deoxyribonucleosides (deoxyadenosine, deoxycytidine (dCyd), azidothymidine, thymidine) and two ribonucleosides (ribopurine, ribouridine (Urd)) on the activity of the two main salvage deoxynucleoside kinases, and on the salvage of dCyd and deoxythymidine (dThd). It turned out that only 2′-deoxythymidine-5′-thiosulphate (dThd-5′-TS) can potentiate the dCK activity, without influencing the thymidine kinase isoenzymes during short-time treatments of human peripheral blood and tonsillar lymphocytes. The enhancement of dCK activity by dThd-5′-TS can be reversed by dCyd, but dThd had no effect on the enzyme activation in cells. Neither dThd-5′-TS nor Urd-5′-TS had any effect on the dCK and thymidine kinase activities tested in cell-free extracts. The stimulation of dCK activity in cells was accompanied by an imbalance in the dThd and dCyd metabolism. The incorporation of 3 H -dThd into DNA was suppressed by 90% in cells by dThd-5′-TS, while Urd-5′-TS only slightly influenced the same process. The 3 H -dCyd incorporation into DNA was inhibited only to 50% of the control, while the 3 H -dCyd labelling of the nucleotide fraction was enlarged in dThd-5′-TS-treated cells, as a consequence of the increased dCK activity. We suggest that the enhancement of dCK activity is a compensatory mechanism in cells that might be induced by different “inhibitors” of DNA synthesis leading to damage of DNA. The increased dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells; this suggestion seems to be supported by the counteracting effect of extracellular dCyd, too.

Collateral sensitivity to gemcitabine (2′,2′-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1–3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2′,2′-difluorodeoxycytidine (gemcitabine, dFdC) and 1-β- d-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-β- d-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl–Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

Stimulation of creatine kinase specific activity in human osteoblast and endometrial cells by estrogens and anti-estrogens and its modulation by calciotropic hormones.

We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17 beta-estradiol (E2) by increased CK specific activity. The effects of E2 on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other antiestrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E2 and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E2. The effects of E2 on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells. Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or human parathyroid hormone (1-34) (hPTH(1-34)) increased the stimulation of CK by E2 by 40-60% relative to E2 alone and significantly increased the sensitivity of the cells to E2 by lowering the effective hormonal dose needed for stimulation of CK by E2 by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)2D3 was due to a 2.5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E2 by hPTH(1-34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)2D3 increased the sensitivity to E2 due to an increase in the number of ER in the cells, whereas PTH(1-34) augmented the response to E2 without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be improved by combined hormone therapy.

Increased deoxycytidine kinase activity by etoposide in L1210 murine leukemic cells.

Deoxycytidine kinase (dCK) is a rate-limiting enzyme for the activation of ara-C. It is responsible for the phosphorylation of ara-C which is widely used in the treatment of leukemia. We examined the effect of etoposide on dCK in L1210 cells and found that incubation with 10 microM etoposide for 1 h increased dCK activity to 170% of the control. This effect of etoposide on dCK activity was concentration-dependent up to at least 100 microM of the substance.

Is serum creatine kinase:aspartate aminotransferase ratio useful for diagnosing acute myocardial infarction in elderly patients?

To evaluate the usefulness of the serum creatine kinase: aspartate aminotransferase (CK:AST) ratio in differentiating between myocardial and non-myocardial increases in serum creatine kinase activity in the elderly. A three month prospective study of all patients admitted to an acute geriatric unit who were clinically assessed and investigated with electrocardiograms and measurement of CK, AST, and lactate dehydrogenase (LDH) activities on the first three days of admission. Excluding those with liver disease and alcohol misuse, patients with increased CK activity were then classified into four groups depending on whether they had fallen or had an acute myocardial infarction (AMI), or both. 270 patients were evaluated. CK activity was raised in 86 (31%) patients on any of the first three days of admission. Of these, 31 had fallen, 19 had an AMI, and five had both fallen and sustained an AMI. The CK:AST ratio, on all days, was higher (p < 0.05) in those who had fallen. On the second and third days, the CK:AST ratio was higher (p < 0.01) in those patients who did not have an AMI. The overlap, however, between these groups was large. These results did not allow discrimination to be made between myocardial and skeletal sources for increased CK activity. The CK:AST ratio is, therefore, of limited use when applied to the diagnosis of AMI in elderly patients. Clinical evaluation rather than the pattern of enzyme change is more likely to determine the cause of increased CK activity.

Effects of downhill running on the responses to an oral glucose challenge.

Because muscle damage from eccentric exercise has been associated with alterations in muscle glycogen metabolism, this study determined the effects of exercise on the insulin and glucose responses to an oral glucose tolerance test (OGTT). In a repeated-measures design, 11 subjects undertook either no exercise, 2 min of isokinetic leg exercise, or 50 min of level or downhill running. No exercise was performed and diet was controlled during the 48 hrs after the treatments and before the OGTT. Ratings of muscle soreness and CK activity were significantly elevated 48 hrs after downhill running. Level running also increased CK activity but did not induce muscle soreness. Isokinetic exercise did not affect either one. Blood glucose responses to the OGTT were similar among the treatments. In contrast, the insulin responses to the OGTT following downhill running were significantly increased. These results suggest that eccentric exercise associated with downhill running that results in delayed muscle soreness is associated with the development of a mild insulin-resistant condition.

Cerebrospinal fluid creatine kinase activity in horses with central nervous system disease: 69 cases (1984-1989)

Summary The csf creatine kinase (ck) activity was determined in 70 csf samples from 69 horses with cns disease. Abnormal values (≥ 1 IU/L) were determined from 32 csf samples, and normal values (&lt;1 IU/L) were found in 38 samples. Increased ck activity was most frequently associated with a diagnosis of equine protozoal myelitis; ck activity was not increased in 11 horses with cervical compressive myelopathy. Other diagnoses, in which csf ck activity was increased included trauma (n = 1), idiopathic epilepsy (n = 2), botulism (n = 2), articular facet fracture (n = 1), intervertebral disk protrusion (n = 1), and toxemia (n = 1).