Enhanced Activity of Deoxycytidine Kinase After Pulsed Low Dose Rate and Single Dose Gamma Irradiation

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<ul><li><p>This article was downloaded by: [Northeastern University]On: 03 December 2014, At: 21:36Publisher: Taylor &amp; FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK</p><p>Nucleosides, Nucleotides and NucleicAcidsPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/lncn20</p><p>Enhanced Activity of DeoxycytidineKinase After Pulsed Low Dose Rate andSingle Dose Gamma IrradiationJ. Sigmond a , J. Haveman b , N. Castro Kreder b , W. J. Loves a , C.van Bree b , N. A. Franken b &amp; G. J. Peters aa Department of Medical Oncology , VU University Medical Center ,Amsterdam, the Netherlandsb Department of Radiotherapy , Academic Medical Center ,Amsterdam, the NetherlandsPublished online: 22 Nov 2006.</p><p>To cite this article: J. Sigmond , J. Haveman , N. Castro Kreder , W. J. Loves , C. van Bree , N. A.Franken &amp; G. J. Peters (2006) Enhanced Activity of Deoxycytidine Kinase After Pulsed Low Dose Rateand Single Dose Gamma Irradiation, Nucleosides, Nucleotides and Nucleic Acids, 25:9-11, 1177-1180,DOI: 10.1080/15257770600894394</p><p>To link to this article: http://dx.doi.org/10.1080/15257770600894394</p><p>PLEASE SCROLL DOWN FOR ARTICLE</p><p>Taylor &amp; Francis makes every effort to ensure the accuracy of all the information (theContent) contained in the publications on our platform. However, Taylor &amp; Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor &amp; Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. 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Terms &amp;</p><p>http://www.tandfonline.com/loi/lncn20http://www.tandfonline.com/action/showCitFormats?doi=10.1080/15257770600894394http://dx.doi.org/10.1080/15257770600894394</p></li><li><p>Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Nor</p><p>thea</p><p>ster</p><p>n U</p><p>nive</p><p>rsity</p><p>] at</p><p> 21:</p><p>36 0</p><p>3 D</p><p>ecem</p><p>ber </p><p>2014</p><p>http://www.tandfonline.com/page/terms-and-conditionshttp://www.tandfonline.com/page/terms-and-conditions</p></li><li><p>Nucleosides, Nucleotides, and Nucleic Acids, 25:11771180, 2006Copyright C Taylor &amp; Francis Group, LLCISSN: 1525-7770 print / 1532-2335 onlineDOI: 10.1080/15257770600894394</p><p>ENHANCED ACTIVITY OF DEOXYCYTIDINE KINASE AFTER PULSEDLOW DOSE RATE AND SINGLE DOSE GAMMA IRRADIATION</p><p>J. Sigmond Department of Medical Oncology, VU University Medical Center,Amsterdam, the Netherlands</p><p>J. Haveman and N. Castro Kreder Department of Radiotherapy, AcademicMedical Center, Amsterdam, the Netherlands</p><p>W. J. Loves Department of Medical Oncology, VU University Medical Center,Amsterdam, the Netherlands</p><p>C. van Bree and N. A. Franken Department of Radiotherapy, Academic MedicalCenter, Amsterdam, the Netherlands</p><p>G. J. Peters Department of Medical Oncology, VU University Medical Center,Amsterdam, the Netherlands</p><p> In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabinepretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the resultof decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase(dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK proteinexpression was observed that indicates a post-translational regulation. The benefit of this increasedactivity induced by irradiation should be further investigated in combination with deoxynucleosideanalogues activated by this enzyme.</p><p>Keywords Radiation; Deoxycytidine kinase</p><p>INTRODUCTION</p><p>Radiation can be given at various schedules (e.g., pulsed low dose rate(LDR) and single dose). As reviewed in Pauwels et al.,[1] gemcitabine(dFdC) pretreatment has been shown to result in significant radiosensiti-zation in both schedules that is most likely due to decreased DNA repair.[2]</p><p>Address correspondence to G. J. Peters, Department of Medical Oncology, VU University MedicalCenter, P.O. Box 7057, 1007 MB, Amsterdam, the Netherlands E-mail: gj.peters@vumc.nl</p><p>1177</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Nor</p><p>thea</p><p>ster</p><p>n U</p><p>nive</p><p>rsity</p><p>] at</p><p> 21:</p><p>36 0</p><p>3 D</p><p>ecem</p><p>ber </p><p>2014</p></li><li><p>1178 J. Sigmond et al.</p><p>Deoxycytidine (dCK) and thymidine kinase (TK) are essential in the synthe-sis of deoxynucleotides required for DNA repair and involved in deoxynu-cleoside analogue activation. Strikingly, dCK also activates deoxynucleosideanalogues such as gemcitabine (dFdC). In vivo, a good correlation betweendCK levels and gemcitabine sensitivity in various murine tumors and humantumor xenografts was observed.[3] Exposure of human lymphocytes to0.52 Gy dosage of -radiation showed an increase in dCK activity.[4] SinceLDR also resulted in an increase in activity of both TK and dCK,[5] weinvestigated whether single dose radiation would lead to a similar effect andtimecourse. We also investigated whether this increase in dCK activity afterirradiation of the human squamous lung carcinoma cell line SW-1573 wasdue to enhanced protein synthesis or post-translational modification whichmight be related to the phosphorylation status of dCK.</p><p>MATERIALS AND METHODS</p><p>The human squamous lung carcinoma cell line SW-1573 was grown inLeibowitz-15 medium as described previously.[5] Cells were irradiated in lateexponential growth (with 5065% of the cells in G1-phase, 3040% in S-phase, 715% in G2-phase as determined by BrdU incorporation).</p><p>LDR irradiation was simulated by giving the cells small irradiation pulsesas described previously.[5] Irradiation was performed followed by a restingperiod of 4 minutes, 52 seconds resulting in a mean dose rate of 1 Gy/h.</p><p>Single high dose irradiation was at a dose rate of 0.6 Gy/min, with 2137Cs sources one above and one below the position of the cell culturedishes. Radioactive assays were performed for measuring dCK activity asearlier described using [3H]-CdA[3] as the substrate. CdA is a very specificdCK substrate and not a substrate for TK2, for example.</p><p>Western blotting by rabbit polyclonal antibodies directed to human dCK(dilution 1:5000) was performed as decribed previously.[6]</p><p>RESULTS AND DISCUSSION</p><p>Both pulsed LDR and single high dose rate irradiation of SW1573increased activities of dCK (Figure 1). The increase after LDR was not onlydose but also time dependent. At the single dose irradiation (4 Gy) thistime dependency was investigated more in detail (Figure 1B) with a plateaubetween 2 and 5 hours. The activity of dCK returned to control levels afterabout 10 hours, possibly because cells were recovered from the effects ofradiation.</p><p>For dCK the increase in activity was not associated with an increasein protein, indicating a posttranslational regulation. This upregulation of</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Nor</p><p>thea</p><p>ster</p><p>n U</p><p>nive</p><p>rsity</p><p>] at</p><p> 21:</p><p>36 0</p><p>3 D</p><p>ecem</p><p>ber </p><p>2014</p></li><li><p>Enhanced Activity of Deoxycytidine Kinase After Radiation 1179</p><p>(Gy)0 10 20</p><p>3</p><p>4</p><p>5</p><p>6</p><p>7</p><p>Time (h)</p><p>En</p><p>zym</p><p>e ac</p><p>tivi</p><p>ty</p><p>0 10 203</p><p>4</p><p>5</p><p>6</p><p>7</p><p>0 10 203</p><p>4</p><p>5</p><p>6</p><p>7</p><p>A B</p><p>6 hours 24 hours</p><p>FIGURE 1 A: dCK activity 6 and 24 h after pulsed LDR irradiation of SW-1573 cells. Enzyme activities areexpressed as percentages of unirradiated controls. Error bars represent SEM of 3 experiments. B: dCKactivity in time after single high dose (4 Gy) irradiation of SW-1573 cells. Enzyme activity is expressed innmol/h/mg protein. The dashed line represents the enzyme activity in unirradiated control cells.</p><p>activity per ng protein after irradiation (Figure 2) is most likely due tophosphorylation. This elevation could be an adaptive response to irradiationand explain the observed increased survival in the initial part of theSW1573 cell survival curve. In literature, there is controversy whetherphosphorylated dCK is active or inactive. Wang and Kucera[7] proposed thatPKC mediated phosphorylation of dCK resulted in a less active form, butthis hypothesis was not confirmed by others.[8] However, cell permeable in-hibitors of PKC did not modify dCK activity in intact cells.[9] Data obtainedwith protein phosphatase clearly indicate that the phosphorylated form ismore active than the dephosphorylated form.[9,10] Thus the hypothesis of arole of PKC in the activation or phosphorylation of dCK is very speculative.We hypothesize that irradiation induces DNA damage, initially leading toincrease of dNTP pools by increasing activity of deoxynucleoside kinases inwhich PKC might be involved.</p><p>2 2 2 2 gy</p><p> gy</p><p> gy</p><p> gy</p><p> 6h6h6h6h</p><p>2 2 2 2 gy</p><p> gy</p><p> gy</p><p> gy</p><p> 24h24h</p><p>24h</p><p>24h</p><p>4 4 4 4 gy</p><p> gy</p><p> gy</p><p> gy</p><p> 6h6h6h6h</p><p>4 4 4 4 gy</p><p> gy</p><p> gy</p><p> gy</p><p> 24 </p><p>h24</p><p> h24</p><p> h24</p><p> h</p><p>6 6 6 6 gy</p><p> gy</p><p> gy</p><p> gy</p><p> 6h6h6h6h</p><p>6 6 6 6 gy</p><p> gy</p><p> gy</p><p> gy</p><p> 24 </p><p>h24</p><p> h24</p><p> h24</p><p> h</p><p>dCK</p><p>dCK</p><p>dCK</p><p>dCK</p><p>----hishis</p><p>his</p><p>his</p><p>c 24</p><p>hc </p><p>24h</p><p>c 24</p><p>hc </p><p>24h</p><p>0</p><p>0,2</p><p>0,4</p><p>0,6</p><p>0,8</p><p>1</p><p>1,2</p><p>1,4</p><p>1,6</p><p>control24 h</p><p>2 Gy 6 h</p><p>2 Gy 24 h</p><p>4 Gy 6 h</p><p>4 Gy 24 h</p><p>6 Gy 6 h</p><p>6 Gy 24 h</p><p>pulsed LDR irradiation</p><p>dC</p><p>K a</p><p>ctiv</p><p>ity </p><p>(act</p><p>ivit</p><p>y/n</p><p>g)</p><p>A B</p><p>FIGURE 2 Western blot (A) of dCK and specific dCK activity per ng dCK protein (B) after pulsed LDRirradiation in SW1573 cells, harvested at different time points. Unirradiated cells at 24 hours (c24h)were used as controls, dCK with a his-tag (dCK-his) was used as a positive control to calculate the amountof dCK protein.</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Nor</p><p>thea</p><p>ster</p><p>n U</p><p>nive</p><p>rsity</p><p>] at</p><p> 21:</p><p>36 0</p><p>3 D</p><p>ecem</p><p>ber </p><p>2014</p></li><li><p>1180 J. Sigmond et al.</p><p>Since antitumor agents activated by dCK such as gemcitabine are usedin combination with radiotherapy in the clinic it might be advantageous toapply these agents during the period of increased enzyme activity inducedby radiotherapy treatment instead of before.</p><p>REFERENCES</p><p>1. Pauwels, B.; Korst, A.E.; Lardon, F.; Vermorken, J.B. Combined modality therapy of gemcitabine andradiation. Oncologist 2005, 10, 3451.</p><p>2. Weiss, C.; Grabenbauer, G.G.; Sauer, R.; Distel, L. Significant increase in residual DNA damage as apossible mechanism of radiosensitization by gemcitabine. Strahlenther Onkol. 2003, 179, 9398.</p><p>3. Kroep, J.R.; Loves, W.J.; van Der Wilt, C.L.; Alvarez, E.; Talianidis, I.; Boven, E.; Braakhuis, B.J.; vanGroeningen, C.J.; Pinedo, H.M.; Peters, G.J. Pretreatment deoxycytidine kinase levels predict in vivogemcitabine sensitivity. Mol. Cancer Ther. 2002, 1, 371376.</p><p>4. Csapo, Z.; Keszler, G.; Safrany, G.; Spasokoukotskaja, T.; Talianidis, I.; Staub, M.; Sasvari-Szekely, M.Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock inhuman lymphocytes. Biochem. Pharmacol. 2003, 65, 20312039.</p><p>5. Castro Kreder, N.; van Bree, C.; Peters, G.J.; Loves, W.J.; Haveman, J. Enhanced levels of deoxycy-tidine kinase and thymidine kinase 1 and 2 after pulsed low dose rate irradiation as an adaptiveresponse to radiation. Oncol. Rep. 2002, 9, 141144.</p><p>6. Hatzis, P.; Al-Madhoon, A.S.; Jullig, M.; Petrakis, T.G.; Eriksson, S.; Talianidis, I. The intracellularlocalization of deoxycytidine kinase. J. Biol. Chem. 1998, 273, 3023930243.</p><p>7. Wang, L.M.; Kucera, G.L. Deoxycytidine kinase is phosphorylated in vitro by protein kinase C alpha.Biochim. Biophys. Acta 1994, 1224, 161167.</p><p>8. Spasokoukotskaja, T.; Csapo, Z.; Sasvari-Szekely, M.; Virga, S.; Talianidis, I.; Eriksson, S.; Staub, M.Effect of phosphorylation on deoxycytidine kinase activity. Adv. Exp. Med. Biol. 2000, 486, 281285.</p><p>9. Smal, C.; Cardoen, S.; Bertrand, L.; Delacauw, A.; Ferrant, A.; Van Den Berghe, G.; Van Den Neste,E.; Bontemps, F. Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid inleukemic cells. Biochem. Pharmacol. 2004, 68, 95103.</p><p>10. Csapo, Z.; Sasvari-Szekely, M.; Spasokoukotskaja, T.; Talianidis, I.; Eriksson, S.; Staub, M. Activationof deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes. Biochem. Pharmacol.2001, 61, 191197.</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Nor</p><p>thea</p><p>ster</p><p>n U</p><p>nive</p><p>rsity</p><p>] at</p><p> 21:</p><p>36 0</p><p>3 D</p><p>ecem</p><p>ber </p><p>2014</p></li></ul>

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