IndianJournal of Biochemistry & Biophysics Vol.42, October 2005, pp. 271-278

CDC27 is involved in radiation response in squamous cell cervix carcinoma

me T Rajkumar'>, G Gopal', G Selvaluxrni" and K R Rajalekshmy'

'Dept. of Molecular Oncology; 2Dept. of Radiation Oncology; 3Dept. of ImrnunologylHematology, Cancer Institute (WIA),Adyar, Chennai 600 020, India

Received 15 March 2005; revised 3 September 2005 sent to In the present study, an attempt was made to identify involved in radiatiorr response in cervix carcinoma. t for the Differential display technique was used to study the expression profiles of tumour biopsy samples obtained from patients, itted for responding and not responding to treatment. The samples were obtained prior to radiotherapy and subsequent to treatment without with Tele-radiation at 10 Gray (Gy). One of the differentially expressed cDNAs, when sequenced was identified to be micating CDC27. Immuno-histochemical analysis of pre- and post-treated tumour samples from fifteen patients showed the down- regulation of expression of CDC27 protein in seven patients. Down-regulation was associated with poorer response to radiotherapy. Cervical cancer cell lines SiHa and C33A were irradiated and their nuclei were stained for expression of CDC27 and analyzed using fluorescent-activated cell sorting (FACS). Down-regulation of CDC27 protein in the irradiated SiHa cell line was associated with greater survival fraction, compared to the irradiated C33A cell line, which had only slight fall in the level of CDC27 protein. This is the first study to suggest a role for CDC27 in radiation response. However, a larger cohort is needed to further confirm the value of CDC27 protein as a predictive marker, for radiation response in cervix cancer.

Keywords: cdc27, cervix cancer, predictive markers, radiation response, differential display, prognosis

)f IPC Code: GOIN33/574 is iction Cervix cancer is the most common cancer among instituted to improve the cure rate. In our earlier study ns; Indian women with a crude incidence rate of of cell cycle regulatory molecules in cervical cancer, 1, 26/100,000 (Madras metropolitan tumour registry c-myc over-expression was found to be an indicator msport, (MMTR), 1998)1. Most of the patients (-80%) are in of poor prognosis in stages II B and III B2. Besides, 3 5 cell stages II B and III B and more than 80% are high- Bcl2 ,4, EGFR , c-erbB2, Ki67 and mitotic index", gradetumours. Squamous cell carcinomas account for MMP2 and TIMP27 were also reported to be the liar nearly 90% of these cancers, while adeno-squamous indicators of poor prognosis in cervical cancer. carcinoma and adenocarcinoma make up the expression analysis can help study radiation- aids, remaining 10%. Although standard radiotherapy treat- induced changes in cells, which could help identify ment is available, nearly 40-60% cases fail the molecules that could act as surrogate markers for treatment. Thus, there is a need for better predictive radiation response and also help predict response to a in the markersthat could identify high-risk women, who are particular modality of treatment. Based on changes in likely to fail the treatment with standard therapy, so gene expression levels and protein function in sbstr, thatadditional chemotherapy/other measures could be response to radiation, genes having roles in various :& cellular process, such as signal transduction'r", cell 'ath, Vet l2 *Corresponding author death 10, DNA repair 11 and cell cycle have been Tel:+91-44-22350340; +91-44-22350241 identified as potential modifiers/markers of radiation entioned Fax:+91-44-24912085 response. E-mail:[email protected] herence. Differential display (DD)i3 has been extensively Abbreviations: DD, differential display; RT-PCR, reverse used to study gene expression to identify differentially 17 transcriptase polymerase chain reaction; FACS, fluorescent- expressed novell4-16 and known genes ,18. A major activated cell sorting; dNTP, deoxy nucleotides; BSA, bovine advantage of this technique is that both tumour serumalbumin; PBS, phosphate-buffered saline; APC, anaphase promotingcomplex. suppressors (which are likely to be down-regulated)

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272 INDIAN J. BJOCHEM. BIOPHYS .• VOL. 42, ocrOBER 2005 and oncogenes (which are likely to be up-regulated) The two tumour biopsy samples obtained from softwar can be identified. Also, the use of relatively small each patient at each time point, were processed as the doi quantities of the sample':' makes it an ideal technique follows - one of the sample was formalin-fixed, the DD to study gene expression changes in tissue biopsy paraffin-embedded and processed for histopatho- GeC ( samples. However, one of the drawbacks of this logical and imrnuno-histochemical evaluation, while as pel procedure is a high rate of false positive candidate the other was snap frozen in liquid nitrogen, imme- reactioi sequences identified. In our recent study on SiHa cell diately after collection. (Prome line, using DD, we identified kDec03 MRPS28 as sequen RNA isolation and RT-PCR being expressed in response to radiation". the G( In the present study, we used DD to identify Total RNA was isolated from biopsy samples using functio differentially expressed genes in tumour biopsy TrizolTM (Life Technologies, Inc., Rockville, MD), according to manufacturers protocol, and was treated samples, obtained from patients who had responded Immune or not responded to the radiotherapy. The tumour with DNase (Amersham Biosciences, UK). RT-PCR IHe for Abl gene c-DNA was performed to check for biopsies were obtained prior to and after 10 Gy pattern DNA contamination. Briefly, 0.2 ug of total RNA was radiation treatment. One of the genes cdc27 was (10 G; reverse transcribed using M-MuLV reverse trans- found differentially expressed and this was confirmed (IS ca criptase (Amersham Biosciences, UK) in 20 III by immuno-histochemical analysis for the CDC27 methor reaction volume containing 200 J.1M dNTP mix, 1 ug protein. Down-regulation of the CDC27 protein was autocls randoin-hexamers (Amersham Biosciences, UK), 2 III associated with poorer response to radiotherapy. (PH 6. SxRT buffer and 1 U ofM-MuLV. The 2 III from the cool t( Materials and Methods reaction was PCR amplified using Abl primers Cell culture incuba (forward primer-5'-GGCCAGTAGCATCTGACTITG- Two human cervical cancer cell lines SiHa and antibot 3'; and reverse primer-S'-ATGGTACCAGGAGTGT- C33A were cultured in Dulbecco's modified Eagle Inc., U TICTCC-3') in a 20 III reaction volume containing medium supplemented with 10% heat-inactivated A cen 10 pmol each of the primers, 200 J.1M of dNTP mix, fetal calf serum, 100 Umrl penicillin, and 100 ug rnI-l found 0.2 U of Taq (Arnersham Biosciences, UK) and 2 III streptomycin, all obtained from Sigma Chemical Co., contro lOxPCR buffer to check for genomic DNA contami- USA. The cultures were maintained at 37°C in air plus was 0 nation. 5% carbon dioxide humidified incubator. scored was I Patients Differential display (DD) Tumoi Fifteen patients, who were diagnosed to have DD was performed using total RNA isolated < treatm cervical cancer, were included in the study, after an from pre- and post-treated (10 Gy) samples of a reactix informed consent. Initial punch biopsy samples were patient who responded and a patient who did not imrmn obtained prior to and after completion of 10 Gray respond to the treatment using DO kits purchased (Gy) dose of tele-radiation treatment. The patients from GenHunter Corp (Nashville, TN), according to CeU irr were treated with external beam radiotherapy using 6 manufacturer's protocol. The anchor and arbitrary Cel MeV linear accelerator (200 cGy/fraction/day/5days a primers that led to the detection of CDC27 accele week/for 5 weeks) and low dose rate brachytherapy were S'-AAGCI IITII II I I"IA-3' and 5'- bolus (dose rate 180-190 cGy/hr). The total dose delivered AAGCTIGA TIGCC-3'. Band isolation and re- cm wr to point A was 70-72 Gy.Brachytherapy was done on amplification was performed as described 13. The re- into tI completion of 50 Gy dose of radiation. In two of the amplified fragment was cloned into a TA cloning descri stage III B patients, who had completed 50 Gy vector (Promega Corp, US) and sequenced using five c treatment, the tumour regression was minimal and M13-21 universal sequencing primer, in a ABI 310 of 2()( intra-cavitary radiotherapy could not be done, as the genetic analyzer. The sequence was submitted to the aroum canal was not defined, hence the whole pelvic dose GenBank database using the Blast-N search function irradi: was taken up to 66 Gy. All the patients were followed for homology search. up until unequivocal evidence of failure or death. All Fluore the failures were found to be due to partial remission, 5' Rapid amplification of cDNA end (5' RACE) Th( followed by progression of disease, which was To further confirm the identity of the sequence, a zation usually observed within 3-6 months after completion S'RACE was performed using SMART RACE kit 18-20 of treatment. (BD Clontech, Palo Alto, CA, US). The OligoTM cells, RAJKUMAR et al.: CDC27 IS INVOLVED IN RADIATION RESPONSE 273 ned from software (Medprobe, Norway) was used to design HEPES pH 7.9, 50 mM NaC!, 0.5 M sucrose, 1 rnM cessed as thedownstream 3' primer based on the sequence of phenylmethylsulfonyl fluoride (PMSF), 0.1 mM alin-fixed, theDD fragment (3' primer- 5'GAG TCC CAA TAT EDTA and 0.5% Triton X-lOO) after incubation for 15 istopatho- GeC CAT TAC 3'). The reaction was performed min on ice with occasional gentle vortexing, pelleted on, while as per manufacturer's protocol. The 5' RACE at 1000 rpm, and re-suspended in ice-cold phosphate- .n, imme- reaction product was cloned into TA cloning vector buffered saline (PBS). The nuclei isolated from the (Promega Corp) and sequenced using universal un-irradiated and irradiated cells were washed once sequencing primers. The sequence was compared with with 1% bovine serum albumin (BSA) in PBS and aliquoted into four tubes each. Two aliquots were iles using the GenBank database using the Blast-N search incubated with 20 III (200 ug/ml) of the anti CDC27 lIe, MD), function. antibody (clone AF3.1, Santa Cruz Biotechnology as treated Immuno-histochemistry (lHC) Inc., USA) and the remaining two aliquots with equal RT-PCR mc evaluation was used to study the expression volume of 1% BSA in PBS at 4°C for 40 min ~heck for pattern of CDC27 in pre-treated and post-treated (negative control). After washing in 1% BSA-PBS, all KNA was (10 Gy) formalin-fixed, paraffin-embedded sections the four aliquotes were treated with FITC-Iabeled se trans- (15 cases) using avidin-biotin peroxidase complex anti-mouse antibody (DAKO) at a dilution of 1:20 '1 20 J..LI method'. Antigen retrieval was performed by and incubated for 30 min at 4°C. After washing in 1% nix, 1 J..Lg autoclaving the sections in 10 mM citrate buffer BSA-PBS, FACS (Becton Dickinson FACSCalibur) JK), 2 J..LI (PH 6.0).The autoclaved sections were allowed to scan was used to analyze the stained nuclei using the from the cool to room temperature for over 20 min and were Becton Dickinson cell quest FACStation software. primers incubated with anti-CDC27 mouse monoclonal The Ml parameter was set for the negative control of lCTTTG- antibody (clone AF3.1, Santa Cruz Biotechnology each cell line at the specified dose (0 or 10 Gy) and f\GTGT- Inc.,USA) at a dilution of 1:20 from 200 ug/ml stock. the same was then applied to the corresponding mtaining A cervix carcinoma tumour tissue section that was antibody-treated sample. The experiment was rTP mix, found to express CDC27 was used as the positive repeated twice. and 2 J..LI control, and for negative control the primary antibody contami- was omitted. T Rajkumar (TR) and G Gopal (GG) CeU survival scored the slides independently and where the scoring Cell survival after exposure to single doses was 6 was not concordant, a joint review was done. assessed by MTT assay as described ". Briefly, 10 cells seeded into 25 em' flasks were exposed to single isolated Tumours were scored without the knowledge of the treatment outcome for the percentage of immuno- .•• dose of 2, 4, 8, 10 Gy, respectively using 6 MeV les of a I X-rays delivered at 200 cGy min- . Experiments were did not reactive tumour cells and the intensity of nuclear conducted to assess the number of cells needed to be irchased immuno-reactivity . seeded into the wells of a 96 well tissue culture plate rding to Cellirradiation so that it does not reach confluence after seven days arbitrary Cells were irradiated using the 6 Me V linear of culture. For both SiHa and C33A, a seeding density CDC27 accelerator (Varian, Palo Alto, CA) with a source to of 1000 cells/well was found to be optimal and this d 5'- bolus distance of 100 em, A field size of 15 cmx25 was used in all further experiments. Irradiated and un- md re- em was used for 175 cm2 flask. The cells were seeded irradiated cells (1000 cells) were plated in a 96 well The re- into the flask and irradiated using 6 MeV X-rays as plate; a minimum of three replicates per dose was cloning described'", A total dose of 10 Gy was delivered in plated. The cells were incubated for 7 days and d using five consecutive daily fractions of 2 Gy at a dose rate assayed using the cell titre'' MTT assay (Promega lEI 310 I of200 cGy min- . The interval between two doses was Corp, USA). Un-irradiated cells were used as controls d to the around 24 hr. The cells from the two cell lines were to calculate the percent survival. The mean values for function irradiated in duplicates, and independently. survival and the standard deviation were obtained from the raw data. The experiment was repeated Fluorescent activated ceUsorting (FACS) twice. The irradiated cells were harvested by trypsuu- rence, a zation along with the un-irradiated cells after approx. Statistical analysis lCE kit 18-20 hr after the final fraction. The nuclei from the Chi squared test with Mantel-Haenszel correction )ligo™ cellswere isolated" in nucleus isolation buffer (10 mM was used to assess the significance and p value. 274 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, OCTOBER 2005

Results The clinical details of the fifteen patients included in the study and the scoring for CDC27 protein expression are given in Table 1. All the patients were treated with standard radical radiotherapy protocol and had provided tumour biopsy samples, prior to treatment and on completion of 10 Gy of radiation dose. In the differential display, more than 30 fragments differentially expressed between the responder and the non-responder were identified (Fig. O. The sequence of one of the differentially expressed band was found to be identical to the mRNA sequence of the human homologue of nuc2 (which is the CDC27 homologue in Schizosaccharomyces pombe), with a score = 452 bits (228), expect = e-124; identities = 2311232 (99%) in the Blast-N search. The sequence was further extended by RACE and was also found to be identical with the human homologue of nuc2 and hCDC27 (expect value = 0.0). Since an antibody to CDC27 that would work on Fig. I-Part of the auto-radiogram ot ':,1edifferential display done formalin-fixed, paraffin-embedded sections was using tumour biopsy samples obtained prior to treatment and after Fig. 2- available (clone AF3.1), immuno-histochemical 10 Gy external beam radiotherapy, from a non-responder to DAB\> respect evaluation was done on the paraffin-embedded treatment and a responder [Lanes 1 and 2, Pre- and post-treated sample, respectively from a non-responder; lanes 3 and 4, pre- and failed t sections from the pre- and the post-treated samples of post-treated sample, respectively from a responder. Pin marks in the fifteen patients. The CDC27 expression was lanes 1 and 4 indicate bands, marked for excision. Arrow denotes showe predominantly found in the nucleus and all the cases the band identified as CDC27] althou positi: Table I-Tumour characteristics, disease status and CDC27 expression strater the fi [CDC27 level in post-treated sample in comparison to the pre-treated sample is given] dow~· S. No. Stage Grade Disease status CDC27 level in post-treated sample (10 G IIIB II Progression - Failed Down-regulated failed 2 IIIB III Disease- free No change expre: cell r IIIB III Progression - Failed No change 3 nucle: 4 IIA III Progression - Failed Down-regulated regula 5 IIIB III Progression - Failed Down-regulated sectio 6 lIB III Progression - Failed Down-regulated chang were 7 IIIB III Progression - Failed Down-regulated Fig. 2 8 IIIB III Progression - Failed Down-regulated expres 9 IIIB II Disease-free Up-regulated respor 10 IIIB III Progression - Failed Down-regulated In ( 11 IIB III Disease-free Up-regulated cancel 12 lIB III Progression - Failed No change exhibi more 13 IIIB III Disease-free No change expres 14 lIB III Progression - Failed No change effect 15 IIIB III Disease-free Up-regulated cells ~ RAJKUMAR et al.: CDC27 IS INVOLVED IN RADIATION RESPONSE 275

ay done nd after Fig.2-Immuno-histochemical staining for CDC27 [Formalin-fixed, paraffin-embedded 5 urn thick sections were used for the study. nder to DAB was used as the colouring reagent and Meyer's hematoxylin as the counterstain [(A and B): Pre- and post-treated tumour sample, -treated respectively, in a patient who responded to treatment; and (C and D): Pre- and post-treated tumour sample, respectively in a patient who ire- and failedtreatment] larks in denotes Dose survi val showed nuclear immuno-reactivity for CDC27, 100 although with varying percentages of tumour cell 80 I-SHa I positivityand intensity. Most of the cases also demon- OC33A

strated weak to moderate cytoplasmic reactivity. Of 0160 the fifteen patients' paired samples, seven showed .~ 40 down-regulation of CDC27 in the post-treated ('/) 'i? (10 Gy) tumour sections and all the seven patients o 20 failed treatment, five did not show alteration in the 0 expression of CDC27 by way of percentage of tumour 2 4 10 cell nuclei that were positive or the intensity of Dose in Gy nuclear immuno-reactivity, and three showed up- Fig. 3-Histogram of cell survival analysis by MTT assay for regulation of CDC27 expression in the post-treated SiHa and C33A' cell lines exposed to single doses of 2, 4, 8 and 10 section. Among the eight cases, who showed no Gy of X rays, respectively [Values are mean of the readings of triplicates measured at 490 run and the error bars represent changeor an up-regulation of CDC27 expression, five standard deviation] were disease-free and three had failed (p = 0.013). Fig. 2 shows the representative examples of CbC27 Using FACS:, ir was found that after treatment with 10 expression in pre- and post-irradiated samples from a Gy radiation, the nuclei of SiHa showed a marked responder (A & B) and a non-responder (C & D). down-regulation of CDC27 expression, with 6% of In order to corroborate the in vivo findings, cervical 'the nuclei expressing CDC27, compared to nearly cancercell lines SiHa and C33A which were shown to 74% of the C33A nuclei expressing CDC27 (Fig. 4). exhibit differential radio-sensitivity (C33A being 23 moresensitive than SiHa ), were studied for CDC27 Discussion expression. Our results also demonstrated a similar The standard prognostic indicators such as stage, effect(Fig. 3). At 10 Gy dose, nearly 25% of the SiHa grade, histologic sub-type win not be useful in our cells survived, compared to _less than 5% of C33A. cervix cancer patients setting, wherein nearly 80% 276 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, OCTOBER 2005

2OO-r------, A The CDC27 protein (APC3), a 823-aa protein, is a B mechani member of the tetratrico peptide repeat (TPR) gene In the CI family", the gene for which has been localized to 25 CDC27 long arm of 17qI2-q21 . It is one of the down-re, eight subunits of the anaphase-promoting complex 6 result in (APCi that is involved in separation of sister contrary chromatids during the transition from metaphase to for idem anaphase of mitosis, probably through ubiquitination dose rad of a centromere protein that regulates the sister study sh chromatid separation process". Thus, the APC plays APC) mJ an important role in mitosis. The other members of regulatioi the APC family include BimE (APCI), CDC16 with H 0 o (APC6), CDC23 (APC8), APC2, APC4, APCS and 2 One t 200~------~ APC726. radiation- The APC is involved in degradation of cyclin B which do during mitosis, which is a pre-requisite for the exit of cycle am the cell from mitosis". Injection of anti-CDC27Hs damage, antibodies into logarithmically growing HeLa cells tumour ce results in cell cycle arrest in metaphase with a normal of CDC2' Fig. 4--FACS analysis of expression of CDC27 protein by human spindle structure/". The APC requires Cdc20 for its their dam cervical carcinoma cells [(A & B): Negative controls of C33A cell activation in mitosis and a Cdc20-related protein The meet line in non-irradiated (A) and after 10 Gy radiation (B); (C & D): CDHl in Gl phase. The expression of Cdc20 is profiles of expression of CDC27 in C33A cell line stained with CDC27 0 monoclonal antibody against CDC27 in non-irradiated (C) and restricted to proliferating cells whereas, the APC and possibility after 10 Gy radiation (D); (E and F): negative controls of SiHa CDHI are expressed in both proliferating and non- the gene c, cell line in non-irradiated (E) and after 10 Gy radiation (F); (G proliferating tissues", The APC (with all its core In conc and H): profiles of expression of CDC27 in SiHa cell line stained subunits) is expressed in several differentiated cells down-regu with monoclonal antibody against CDC27 in non-irradiated (G) including the terminally differentiated neurons and is and after 10 Gy radiation (H). A to H are representative profiles of 3o poorer res one of the two measurements in one experiment] tightly associated with CDH1 . The APC-CDHl view of tl complex was shown to have a high cyclin B value of C were in stages II B and III B, with more than 80% ubiquitination activity In mitosis and as yet other roles radio-respc being high grade tumours and more than 90% being in the G 1 phase of cell cycle". evaluated r squamous cell carcinomas. Most of our patients were The human homologue of fission yeast CDC27 has treated with radiotherapy in view of their locally also been shown to be a functionally important Acknowle< 32 advanced nature of disease. Predictive molecular subunit of DNA polymerase delta ,33 aiding in the This stuc markers for radiation response can be very useful in interaction of DNA polymerase delta with the of Atomic identifying those patients who are at high risk for processivrty factor proliferating cell nuclear antigen 310 genetic failure following radiotherapy. Identification of this (PCNA), which is essential for effective replication of Chennai W high-risk subset of patients can help introduce the genome. Thus, it appears that CDC27 protein may We would additional modalities such as chemotherapy for play a role in cell cycle regulation through several Research l treatment. mechanisms. Hammersmi comments 0 The present study showed that in cervix cancer Fas-induced activation of cdk's in Jurkat cells is patients CDC27 levels may be altered in response to shown to be due to caspase-3-mediated destruction of References radiation and the down-regulation of the CDC27 Weel (an inhibitory kinase of cdc2 and cdk2) and I Shanta V, levels is associated with a poorer outcome to CDC27, resulting in a loss of APC function, leading incidence radiotherapy. The in vitro study using cell lines SiHa to inhibition of cyclin degradatiorr'", In spite of the 1984-199l and C33A also corroborated the in vivo data, in as increased cdk activity during Fas-induced apoptosis in Cancer Ins 2 Vijayalah much as the less radio-sensitive cell line SiHa Jurkat cells, mitosis did not occur. In endothelial cells, (2002) EU/ KlPl showing a marked down-regulation of CDC27, after caspase-mediated destruction of p21 CIPl and p27 3 Rajkumar exposure to lOGy radiation, compared to the radio- results in apoptosis ". However, in Jurkat cells it is not A & Shant sensitive C33A (Figs 3 and 4). seerr'", indicating that there may be different 4 Pillai M R Res ClinO RAJKUMAR et al.: CDC27 IS INVOLVED IN RADIA nON RESPONSE 277 protein, is a mechanisms triggering apoptosis in different systems. 5 Kersemaekers A M, Fleuren G J, Kenter G G, Van den Broek :rPR) gene In the cervical tumour cells, the down-regulation of LJCM, Uljee S M, Hermans J & Van de Vijver M J (1999) ocalized to Clin Cancer Res 5, 577-586 CDC27may not be associated with this pathway, as s one of the 6 Nakano T, Oka K, Ishikawa A & Morita S (1998) Cancer down-regulation could lead to apoptosis, which might Ig complex Detect Prev 22, 120-128 result in better response to radiotherapy, which is 7 Davidson B, Goldberg I, Kopolovic J, Lerner-Geva L, I of sister contraryto our findings. Zhou and Rigaud'" used DD Gotlieb W H, Ben-Baruch G & Reich R (1999) Gynecol etaphase to for identifying changes in gene expression to low Oncol73, 372-382 iquitination doseradiation (4 Gy) in human lymphoblasts. The 8 Jung M & Dritschilo A (1996) Seminars Radiat Oncol 6, the sister study showed that Cdc16 (another subunit of the 268-272 APC plays 9 Harari PM & Huang S M (2001) Seminars Radiat Oncol 11, APC)mRNA was down-regulated. A similar down- nembers of 281-289 regulation was also observed after oxidative stress 10 Friedman A H, KoIesnic R N & Fuks Z (1996) Seminars ), CDCl6 36 withH 0 . APC5 and 2 2 Radiat Oncol6, 273-283 One hypothesis we propose is that, following 11 Leadon S (1996) Seminars Radiat Oncol6, 295-305 radiation-induced DNA damage, in tumour cells 12 Rudoltz M S, Kao G, Blank K R, Muschel R J & McKenna If cyclin B whichdown-regulate CDC27, there could be a cell WG (1996) Seminars Radiat Oncol6, 284-294 the exit of cycle arrest/delay, which could allow repair of the 13 Liang P & Pardee A B (1992) Science 257, 967-97l -CDC27Hs damage, enabling them to survive. Conversely, 14 Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer W HeLa cells E, Vande Woude G F, O'Connor P M & Appella E (1997) tumourcells, which have not shown down-regulation Proc Natl Acad Sci (USA) 94, 6048-6053 h a normal of CDC27, may continue to cycle without repairing 15 Arakawa H, Nakamura T, Zhadanov A B, Fidanza V, Yano c20 for its theirdamaged DNA, which could trigger apoptosis. T, Bullrich F, Shimizu M, Blechman J, Mazo A, Canaani E ed protein The mechanism, by which the down-regulation of & Croce C M (1998) Proc Natl Acad Sci (USA) 95, 4573- Cdc20 is CDC27 occurs in the tumours is not known. The 4578 e APC and possibilityof DNA methylation-mediated silencing of 16 Thrash-Bingam C A & Tartof K D (1999) J Natl Cancer Inst 91, 143-151 ~ and non- thegene cannot be ruled out. 11 its core 17 Lawlor E R, Lim J F, Tao W, Poremba C, Chow C J, In conclusion, we have shown that CDC27 when Kalousek I V, Kovar H, MacDonald T J & Sorensen PH B :iated cells down-regulated in cervix cancers is associated with (1998) Cancer Res 58, 2469-2476 .ons and is poorerresponse to radiation treatment. However, in 18 Hisamatsu T, Watanabe M, Ogata H, Ezaki T, Hozawa S, ,PC-CDHI view of the small number of patients studied, the Ishii H K, Anai T & Hibi T (1999) Cancer Res 59, cyclin B valueof CDC27 protein as a predictive marker for 5927-5931 other roles 19 Gopal G & Rajkumar T (2005) Indian J Biochem Biophys radio-response in cervix cancer needs to be further 42,81-86 evaluatedprospectively in a larger patient population. 20 Scott S L, Earle J D & Gumerlock P H (2003) Cancer Res :DC27 has 63,7190-7l96 Acknowledgement important 21 Fish K N, Naueler C, Mills L, Stephanstenglein & Nelson J ing in the This study was supported by a grant from the Dept. A (1998) Virol72, 5661-5668 with the of Atomic Energy, Government of India. 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