Radiosensitivity Prediction

PartⅠPrediction of radiation sensitivity of nasopharyngeal cancerIntroductionChina is one of the countries in the world where the morbidity of nasopha-ryngeal cancer(NPC)is high,radiation therapy is a main treatment method of NPC.Radiation sensitivity is different among all patents with NPC,and which can affect the NPC radiation curative rate markedly.Thus,to predict the radia-tion sensitivity of NPC exactly is very important,but so far,effective index of which hasn't been found yet.testⅠthe relationship between radiation sensitivity and oncogene expression of NPCrecently,it has been reported that the radiosensitivity of oncogene-trans-fetted cells was related with expression degree of oncogene.But there hasn't study report by now on the correlativity of abnormal p53 protein and the expres-sion degree of bcl-2 gene with NPC radiosensitivity.This test measured the ab-normal p53 protein and the expression degree of bcl-2 gene in NPC tissues with the technique of immunohistochemical staining,and analyzed the relationship of which with the radiation sensitivity of cervical metastatic node areas of NPC pa-tients.Materials and methods1.cases61 NPC patents with cervical nodal metastasis who underwent radiation therapy initially at no.1 hospital of china medical university between January 2000 and January 2002 were analyzed,all of the cases had nasopharyngeal his-topathological confirmation and were approved poorly differentiated squamous cell carcinoma.2.radiation therapyall patients received external radiation using 6-MV linear accelerator (LINAC).Tumor doses of nasopharynx ranged from 70 to 74 Gy in 35-37 fractions with a period of 7-8 weeks;while the nodal target doses of cervical metastatic area were 66-70 Gy in 33-35 fractions,6-7 weeks.3.measurement of target volumea special clinican measured the largest cervical metastic lymph nodes every day to obtain the largest diameter(a)and perpendicular value(b),and figured out their volume according to formula;node volume=ab2/2.4.immunohistochemical stainingthe pathological slices of all cases were stained with routine HE method and got immunohistochemical staining for abnormal p53 protein as well as bcl-2 gene.SP method was adopted during immunohistochemical staining.5.reading slicesto read staining results by a special person with double blind method and calculate the ratio of positive cells to total tumor cells number;definite the cases with ratio of 0,1-25%,26-50%,51-75%,＞75%as-,+,+ +,+ + +,+ + + + respectively.6.statistical managementall data were dealt with SPSS10.0 software,and compared about every group average with T test,the relativity were analyzed by Spearman rank correl-ative analysis.ResultsThe relationship between there oncogene expression degree and reduction of cervical metastatic lymph nodes;We determined the irradiation dose which were required to reduce the nodal volume by 25%,50%,75%and 100%as radiosensitivity during the treatment period,that is,the lower the dose required,the higher the radiosensitivity was. According to Spearman rank correlative analysis,abnormal P53protein and the expression degree of bcl-2 was positively related with the total dose that was re-quired for reduction of cervical metastatic lymph nodes(P＜0.05),and negative related with radiosensity.In addition,the content of abnormal P53 protein had nothing with the ex-pression degree of bcl-2 gene(correlative factor was 0.201,P＞0.05).DiscussionThis study suggested that abnormal P53 protein content and bcl-2 gene ex-pression degree was negative related with radiosensitivity of cervical metastatic lymph nodes in NPC patients(P＜0.05),there wasn't correlativity between ab-normal P53 protein content and bcl-2 gene expression(P＞0.05).Abnormal P53 protein properly affected tumor radiosensitivity by ways as follows;(1)abnormal P53protein interfered G1 phase inhibition of DNA-dam-aged cells,and made them go into S phase continously.(2)When plenty of was injuried after radiation therapy,abnormal P53 protein could reduce the cell apoptosis.Bcl-2 gene might affect tumor cells byreducing radation-induced cell apoptosis.Test-ⅡThe relationship between radiosensitivity and cyclins content of NPC.It was reported that some cyclins might have beating on the prognosis and intrinsic radiosensitivity of some kinds of tumorsm.There haven't reports about the relationship between cyclins and radiosensitivity of NPC by now.This test measured content of cyclins PCNA,ki-67 and cyclin-Bi by immuno-histo-chemical staining of method and analyzed its influence or radiosensitivity of cer-vical metastatic lymph nodes in NPC.Materials and methods1.Case.It was the same as test 1. 2.Radioationtherepy.It was the same as test 1.3.Measurement of targent volume.It was the same as test 1.4.Immunohistochemical staining.All pathological slices were stained by noutine HE method as well as immunohistochemical method for PCNA,ki-67 and cyclin B1.SP method was used durning immuno-histoehemical steining.5.Reading slices.It was the same as test 1.6.Statistical management.It was the same as test 1.ResultRelationship between cyclins content and reduction of cervical metastatic lymph nodes.We determined the irradiation dose which were required to reduce the nodal volume by 25%,50%,75%and 100%as radiosensitivity during the treatment period,that is,the lower the dose required,the higher the radiosensi-tivity was.Cyclins PCNA and ki-67 content was negatively related with total radiation dose required during the reduction of cervical metastaic lymph nodes (P＜0.05),that is,it was positively related with radiosensitivity,while the content of cyclin B1 had no relativity with the radiation dose required during re-duction of cervical target volume(P＞0.05).In addition,content of cyclin PC-NA and ki-67 was positively related,(correlative factor was 0.243,P＜0.05); Both of them had nothing with the content of cyclin B1(correlative factor was 0. 164 and 0.098 respectively,P>0.05).DiscussionThis study suggested that the radiosensitivity of cervical metastatic area was different if the content of PCNA in nasopharyngeal cancer tissue,and they were in positive relationship.This might be because PCNA as a main label of cell proliferation was definitely related with cell proliferation rate,while the latter was positively related with radiosensitivity.During this study,content of cyclin ki-67 was positively related with radi-osensitivity of cervical metastatic lymph nodes,in coincidence with the regulari-ty that the radiosensitivity of hyperproliferative tumor was high. ConclusionsThis study found that abnormal P53 protein bcl-2 gene expression degree in NPC tissue was negatively related with radiosensitivity cervical metastatic are-as,and content of PCNA and ki-67 was positively related with radiosensitivity of cervical metastatic areas,but cyclin B1 had nothing with it.Owing to cervical metastatic lymphanodes and primary lesions were homologous in pathological type,abnormal P53 protein,bcl-2 gene expression degree,content of PCNA, ki-67 could predict radiation sensitivity of NPC,with high accuracy superior to the method by which radiation sensitivity was determined based on tumor differ-entiation degree.PartⅡRadiation enhanced sensitivity effect of recombinated human interferonα-2a on human nasopharyngeal cancer cell series CNE-1IntroductionRecently,interferonαhas not only tumor cytotoxity but also the effect of inducing tumor cells apoptosis and modulating proliferation cycle of tumor cells. Because radiation sensitivity of tumor cells was closely related with distribution of cells in proliferation cycle,interferonαmight change radiation sensitivity of tumor cells,There hasn't report by now about that interferon can affluence of recombinated human interferonα-2a on NPC cells series CEN-1 using cellu-lar clone formation method.Materials1.Cell culture.Human NPC cells series CNE-12.Drug.Recombinated interferonα-2a(IFN a-2a).to repare culture fluid(PRMI1640)with suitable concerntration.(0.5×10~31u/ml,1.0×1031u/ ml,1.5×1031u/ml)before test.3.Major equipment.FACS can flouing cytomrtry,carbon dioxide culture box,upset microscope,linear accelerator.Methods1.Influence of IFNα-2a on radiosensitivity of CNE-1 cells;We digested CEN-1 cells proliferating with logarithm in culture bottle u-sing pancreatin,calculated living cells,diluted by grade and inoculated on 6cm plate.The cases were divided into four groups;(1)Control group;no treatment at all;(2)Simply administration group;added 5ml culture matrix containing IFNα-2a with a concentration of 0.5×10~3IU/ml,1.0×10~3IU/ml,1.5×10~3IU/ml respectively,cultured for 24hrs and discarded drugs,then cultured continously for 14 days;(3)Simply radiation group;radiated the cells with a dose of 1,3,5,7,9 ray using 6my×ray,then cultured 24 hours after affected by IFNα-2a with different concentration,conditions were the same as(2),(3).We calculated cellular clones 14 days after cell culture,figured out cellular survival rate and drew cellular survival curve using SPSS 10.0 software.We ca-culated radiation enhanced sensitivity ratio of IFNα-2a on cellular survival curve at a leval of 10%survival rate.2.Influence of IFNα-2a on CEN-1 cellular proliferation cycle;We digested CEN-1 cells proliferating with logarithm in culture bottle u-sing pancreatin.All case were divided into two groups;(1)control group;no treatment at all;(2)administration group;added 5ml culture fluid containing IFNα-2a with concentration of 1.0×10~3IU/ml,cultured 24 hrs and abandoned up drug, then cultured continuously.Chose cells in control group and administration group cultured after 0,24 and 48 hrs,then digested them and made single cellu-lar suspension.Detection Of DNA content;testified DNA content in CNE-1 cells with flowing cytometry and figured cellular distribution of every phase in cellular cy-cle. Result1.Influence of IFNα-2a on radiation sensitivity of CNE-1 ceils.According to cellular relative survival curve,we detected SER 24 hrs after IFNα-2a action,and results of were 1.16,1.61,1.85 with the concentration of 0.5×10~3IU/ml,1.0×103IU/ml×1.5×10~3IU/ml respectively at a cellular survival leval of 10%.2.influence of IFNa-2a on CNE-1 cellular proliferation cycle.CNE-1 cell cycle time(46.6h)was increased longer than control group (38.8h)24hrs after acted by IFNα-2a by CNE-1(P＜0.05),of the total, the prolongation of G1 phase(23.3h)and the shortening of S phase(4.2hrs) was comparable significantly with control group(17.6h and 6.9h)(P<0.05). The change of every phase in CNE-1 cell cycle wasn't obvious 48hrs after trea-ted by IFNα-2a compared with control group(P>0.05).DiscussionIn the study,S phase time of CNE-1 cells in proliferation cycle was shor-ter 24hrs after acted by 1.0×10~3IU/ml IFNα-2a than control with statistical significance.We also found that survival curve shape of human NPC CNE-1 cells after radiation changed with increase of IFNα-2a concentration,increase of oblique rate of linear part suggested that IFNα-2a had somewhat enhanced radiation effect and was concentration dependent.It might action of IFNαon CNE-1 cells induced S phase decrease in cytocycle and made radiation resistant S phase cells decrease,then increased radiation sensitivity of CNE-1 cells consequently.Interferon could lead S phase term a bridge in proliferation cycle of NPC cells,and enhance the radiation sensitivity of NPC,that might offer a theorecti-cal foundation for the application of interferon during the general treatment with radiation therapy. ConclusionsHuman recombinated interferonα-2a had enhanced radiosensitive effect on human NPC cells CNE-1;human recombinated interferonα-2a could lead prolongation of G1 phase and abridge of S phase of human NPC cells CNE-1.