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Protein expression following gamma-irradiation relevant to growth arrest and apoptosis in colon cancer cells with mutant p53
Linköping University, Department of Clinical and Experimental Medicine, Oncology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Oncology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Oncology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
2009 (English)In: Journal of Cancer Research and Clinical Oncology, ISSN 0171-5216, E-ISSN 1432-1335, Vol. 135, no 11, 1583-1592 p.Article in journal (Refereed) Published
Abstract [en]

We previously found that p53, p73, survivin and PRL were implicated in the outcome of radiotherapy in rectal cancer patients. In the present study, we tried to understand mechanisms of colon cancer cell line response to radiation based on protein expression related to proliferation and apoptosis. KM12C, KM12SM and KM12L4a, cell lines with one origin, were radiated with 0, 10 or 15 Gy γ-radiation. Radiosensitivity was determined with cell cycle and apoptosis analysis, and protein expression of TAp73, ΔNp73, mutated p53, survivin and PRL-3 was determined by Western blot. KM12C showed transient G2-arrest, low apoptosis and up-regulation of resistance factors such as PRL-3. In KM12C expression of ΔNp73 increased after 10Gy, but not after 15Gy. KM12SM had permanent G2-arrest, low apoptosis and showed up-regulation of the anti-apoptotic survivin and down-regulation of the pro-apoptotic TAp73 and the radioresistance factor PRL-3 was down-regulated. KM12L4a, the most radiosensitive cell line, showed up-regulation of TAp73 and down-regulation/no up-regulation of resistance factors such as ΔNp73, survivin and PRL-3 after radiation. In conclusion, the KM12C cell line was more radioresistant than KM12L4a regarding apoptosis and certain apoptotic proteins. The radiosensitivity of KM12L4a might partly depend on the lack of up-regulation of proteins negative for the outcome of radiotherapy.

 

Place, publisher, year, edition, pages
2009. Vol. 135, no 11, 1583-1592 p.
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:liu:diva-14786DOI: 10.1007/s00432-009-0606-4OAI: oai:DiVA.org:liu-14786DiVA: diva2:25265
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2017-12-13Bibliographically approved
In thesis
1. p73 in colorectal cancer
Open this publication in new window or tab >>p73 in colorectal cancer
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Colorectal cancer (CRC) is the third most common cancer in the world, with about 5000 new cases in Sweden every year. CRC is caused by mutation (inherited or acquired) in genes, by gene variants and changed expression of proteins. The primary way to achieve a curative result for CRC is to remove the tumor by surgery. To reduce risk of recurrence chemo- or radiotherapy are given as a complement to surgery. p73 is a structural and functional homologue of tumor suppressor p53. However, p73 is rarely mutated in tumors, but rather overexpressed as compared to normal tissue. There are two main isoforms of p73, the transactivation capable TAp73 and the truncated ΔNp73, which are involved in an autoregulatory loop with TAp73 and p53.

The aim of this study was to investigate the role of p73 and related proteins in the development and treatment of CRC. A G4C14-to-A4T14 polymorphism of p73 was studied in CRC patients and healthy controls (Paper I), and rectal cancer patients who were randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper II). The AT/AT genotype of the p73 polymorphism may increase risk of CRC development and CRC patients with the AT allele had a better prognosis. When dividing the cases into colon and rectal cancer it was seen that in colon cancer the AT allele tended to be more favorable for overall survival, while in rectal cancer the GC allele seemed to be more favorable. Rectal cancer patients, with a combination of GC/GC genotype, wild type p53 and weak survivin expression survived longer after preoperative radiotherapy. This was not observed in the patients only receiving surgery. The protein expression of p73 was further studied in the rectal cancer patients randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper III). p73 was expressed higher in tumor tissue than in normal mucosa. Patients with p73 negative tumors had a lower risk of local recurrence after radiotherapy, as opposed to patients that had p73 positive tumors or patients with p73 negative tumors that did not receive radiotherapy. Effects of γ-radiation was further studied in colon cancer cell lines KM12C, KM12SM and KM12L4a regarding cell cycle, survival fraction (clonogenicity), apoptosis and protein expression patterns of mutated p53, TAp73, ΔNp73, survivin and PRL-3 (Paper IV). KM12C displayed low survival fraction, low apoptosis, no cell cycle arrest and an upregulation of the antiapoptotic ΔNp73 after irradiation. KM12L4a showed a high survival fraction, but high apoptosis, arresting of the cell cycle and upregulation of the radio-resistance factor survivin. The effects of overexpression and knockdown of survivin on TAp73, ΔNp73 and p53 expression in colon cancer cell lines HCT-116p53+/+ and HCT-116p53-/- with and without γ-radiation were studied (Paper V). Overexpression of survivin decreased wild type p53, whilst downregulation of survivin lead to a simultaneous downregulation of TAp73 and ΔNp73, mRNA and protein, both with and without γ- radiation. Knockdown of survivin also demonstrated an increase in apoptosis.

In conclusion, we showed that the G4C14-to-A4T14 polymorphism of p73 and p73 protein expression may be involved in CRC development, radiotherapy response and survival. We further showed that TAp73, ΔNp73 and p53 were regulated by survivin in colon cancer cells.

Abstract [sv]

Cancer i tjocktarmen (kolon) och ändtarmen (rectum) är den tredje vanligaste cancerformen i världen och i Sverige drabbas varje år ca 5000 personer av tjockoch ändtarmscancer. Antalet drabbade individer är högre i de industrialiserade länderna än övriga världen, vilket tyder på att vissa omgivnings- och livsstilsfaktorer påverkar risken för tjock- och ändtarmscancer. Även vissa genetiska faktorer påverkar risken för insjuknande i dessa cancerformer.

En gen är mallen för ett eller flera proteiner och en förändring (mutation) i en gen kan innebära att motsvarande protein förändrar eller förlorar sin normala funktion. En del gener har också nedärvda naturliga variationer, så kallade polymorfier, vilka i sin tur kan leda till variation i funktionen hos motsvarande protein. Proteiner som förändrat eller förlorat sin normala funktion bidrar till de speciella egenskaper som finns hos cancerceller. Cancerceller till skillnad från normala celler delar sig okontrollerat, är motståndskraftiga mot den programmerade celldöd (apoptos) som normalt förstör skadade celler och kan sprida sig via blodbanan till andra organ (metastasera).

Tjock- och ändtarmscancer behandlas främst med kirurgi, cellgifter och strålning och tack vare förbättrade behandlingar har dödligheten minskat de senaste årtiondena. Genom att studera olika genetiska varianter, proteiner och förhållandet mellan proteiner i cancerceller får vi större insikt i vilka faktorer som ökar risken för tjock- och ändtarmscancer, men också vilka faktorer som påverkar hur effektiv en behandling är för varje individuell patient.

Målet med denna avhandling var att studera proteinet p73 och hur den påverkar risken för tjock- och ändtarmscancer och behandlingseffekten av sjukdomen. p73 tillhör samma proteinfamilj som “genomets väktare” p53 och båda har förmågan att skydda cellen genom att stoppa celldelning och inducera apoptos i skadade celler. Dock är p53 muterat i ungefär 50 % av alla tjock- och ändtarmscancrar.

Genom att studera en polymorfi i p73 hos tjock- och ändtarmscancerpatienter och friska blodgivare såg vi att de individer som har dubbel uppsättning av variantgenen har ökad risk att utveckla tjock- och ändtarmscancer och att de patienter som är bärare av variantgenen hade en längre överlevnad (Paper I). Ändtarmscancerpatienter som fått strålbehandling överlevde längre om de hade dubbel uppsättning av den ursprungliga p73-genen, icke-muterat p53 och lågt uttryck (mängd) av survivin. Survivin är ett protein som normalt hindrar cancerceller från att dö (Paper II). De ändtarmscancerpatienter som behandlats med strålning och som hade p73 negativa tumörer fick mycket färre återfall än de patienter som hade p73 negativa tumörer men inte fått strålbehandling, eller som hade p73 positiva tumörer (Paper III). För att närmare studera tjocktarmscancercellers motståndsmekanismer mot strålbehandling mättes bland annat olika typer av celldöd och uttrycket av två olika varianter av p73, kallade TAp73 och ΔNp73, survivin och PRL-3. PRL-3 är ett protein som uttrycks starkast i metastatiska tumörer och leder till ökat motstånd mot strålbehandling. Den cellinje som inte dog genom apoptos hade högt uttryck av ΔNp73, vilket är en variant av p73 som hindrar celler från att dö, medan den som trots strålning i hög grad fortsatte dela sig hade ett högt uttryck av survivin (Paper IV). Efter indikationer om att uttrycket av p73 proteinerna TAp73 och ΔNp73 är kopplade till survivin undersöktes ett eventuellt förhållande mellan dessa proteiner i tjocktarmscancerceller. I de fall där uttrycket av survivin minskades i cellerna minskade också uttrycket av TAp73 och ΔNp73. Detta fenomen sågs även i celler som behandlats med strålning. En minskning av survivin i cellerna ledde även till att fler celler dog genom apoptos (Paper V).

Sammanfattningsvis pekar våra resultat på att p73 spelar en viss roll i utvecklingen av tjock- och ändtarmscancer, men också avseende effekterna på strålbehandling av ändtarmscancer. Vi har också visat att TAp73, ΔNp73 och p53 regleras av survivin.

 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 99 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1108
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18432 (URN)978-91-7393-677-4 (ISBN)
Public defence
2009-05-29, Eken, Hälsouniversitet, ingång 65, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-05-26 Created: 2009-05-26 Last updated: 2009-08-21Bibliographically approved
2. Molecular Studies of Irradiation and SN-38 on Colorectal Cancer
Open this publication in new window or tab >>Molecular Studies of Irradiation and SN-38 on Colorectal Cancer
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Colorectal cancer (CRC) is one of most common cancer diseases worldwide. In Swedenapproximately 5,000 new cases of CRC are generated each year, which makes it the thirdmost common cancer disease among both men and women. The past decades ofimproved treatment strategies have considerably increased the five-year survival for CRCpatients. However more could be achieved in this area, in particular for metastatic CRC,which is the cause of most CRC-related deaths. Therefore it is important to study thebiological response to certain treatments induced in CRC to find valuable predictiveand/or prognostic factors to select patients for better suited treatments.

The aim of this thesis was to gain insight into the molecular changes that occurfollowing irradiation or treatment with SN-38 in rectal cancer patients or colon cancercell lines by studying the RNA expression, protein expression, DNA cell cycledistribution and apoptotic response. The expression of phosphatase of regenerating liver(PRL) proteins was investigated in rectal cancers from 125 patients included in arandomized clinical trial of preoperative radiotherapy (RT). Increased expression of PRLswas seen at the invasive margin of primary and metastatic cancers compared with theinner area of the tumors. Moreover, strong PRL staining at the invasive margin correlatedto distant recurrence and worse survival of patients in the RT group but not in non-RTgroup (Paper I). Radiosensitivity was studied by treating KM12C, KM12SM andKM12L4a colon cancer cell lines with radiation. KM12C is of low metastatic naturecompared with the highly metastatic KM12SM and KM12L4a. Upregulation of ΔNp73and PRL-3 might contribute to the radioresistant phenotype in KM12C. In contrast,KM12L4a shows a high frequency of apoptosis and lack of upregulation of ΔNp73, PRL-3 and survivin, which might explain its radiosensitive phenotype (Paper II). KM12C,KM12SM and KM12L4a were treated with SN-38 which inhibits topoisomerase 1 (topo-1). The results show that SN-38 induces G2/S arrest and possess the capacity to triggerapoptosis in the three cell lines (Paper III). To further elucidate SN-38 effect on these celllines, the gene expression profile following SN-38 treatment was studied. Oligonucleotidearrays consisting of ~27,000 spots were hybridized with sample and reference cDNA.Both unsupervised and supervised hierarchical clustering analysis, and functional analysiswere performed. Supervised hierarchical clustering gives a strong signal of 1453discriminated genes, the vast majority being upregulated. Both upregulated anddownregulated genes point toward a favorable impact of SN-38 regarding the apoptoticpathways. For example RhoB and Bax are upregulated together with downregulation ofKras and survivin, which promotes apoptosis (Paper IV).

In conclusion, PRLs may be valuable biomarkers for RT resistance, predicting apoor prognosis in rectal cancer patients. Targeting radio-resistance factors, such asΔNp73 and survivin may contribute to an increased sensitivity to RT. SN-38 affects cellproliferation and apoptosis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 60 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1070
Keyword
Colorectal cancer (CRC), molecular changes, SN-38
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-14789 (URN)978-91-7393-838-9 (ISBN)
Public defence
2008-09-29, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2009-08-22Bibliographically approved

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Pfeifer, DaniellaWallin, ÅsaHolmlund, BirgittaSun, Xiao-Feng

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