Expression of DNA-damage response and repair genes after exposure to DNA-damaging agents in isogenic head and neck cells with altered radiosensitivity

Background: Increased radioresistance due to previous irradiation or radiosensitivity due to human papilloma virus (HPV) infection can be observed in head and neck squamous cell carcinoma (HNSCC). The DNA-damage response of cells after exposure to DNA-damaging agents plays a crucial role in determining the fate of exposed cells. Tightly regulated and interconnected signaling networks are activated to detect, signal the presence of and repair the DNA damage. Novel therapies targeting the DNA-damage response are emerginghowever, an improved understanding of the complex signaling networks involved in tumor radioresistance and radiosensitivity is needed. Materials and methods: In this study, we exposed isogenic human HNSCC cell lines with altered radiosensitivity to DNA-damaging agents: radiation, cisplatin and bleomycin. We investigated transcriptional alterations in the DNA-damage response by using a pathway-focused panel and reverse-transcription quantitative PCR. Results: In general, the isogenic cell lines with altered radiosensitivity significantly differed from one another in the expression of genes involved in the DNA-damage response. The radiosensitive (HPV-positive) cells showed overall decreases in the expression levels of the studied genes. In parental cells, upregulation of DNA-damage signaling and repair genes was observed following exposure to DNA-damaging agents, especially radiation. In contrast, radioresistant cells exhibited a distinct pattern of gene downregulation after exposure to cisplatin, whereas the levels in parental cells were unchanged. Exposure of radioresistant cells to bleomycin did not significantly affect the expression of DNA-damage signaling and repair genes. Conclusions: Our analysis identified several possible targets: NBN, XRCC3, ATR, GADD45A and XPA. These putative targets should be studied and potentially exploited for sensibilization to ionizing radiation and/or cisplatin in HNSCC. The use of predesigned panels of DNA-damage signaling and repair genes proved to offer a convenient and quick approach to identify possible therapeutic targets

Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity

. Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods. Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assaypost irradiation cell cycle distribution was studied by flow cytometrythe expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results. Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions. We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRSpositive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints

Radiation therapy dose adjustment in human papillomavirus associated squamous cell carcinoma of the oropharynx in vitro and in vivo

Znanstveno izhodišče: Humani virus papiloma (HPV) je eden pomembnejših povzročiteljev ploščatoceličnega karcinoma ustnega žrela. Opazili so, da HPV-pozitivni ploščatocelični karcinomi ustnega žrela bolje odgovorijo na sočasno radiokemoterapijo s terapevtiki na osnovi platine od HPV-negativnih ploščatoceličnih karcinomov ustnega žrela. To je vzbudilo razmišljanje o znižanju intenzivnosti radioterapije pri teh bolnikih. Pogoj je ohranitev deleža ozdravitev, ki je dosežen s konvencionalnimi dozami obsevanja (frakcionirano obsevanje, skupna doza obsevanja 70 Gy) in sistemskih terapevtikov, namen pa zmanjšanje neželenih stranskih učinkov zdravljenja. Do sedaj opravljene raziskave kažejo, da so boljši rezultati zdravljenja HPV-pozitivnih ploščatoceličnih karcinomov ustnega žrela posledica večje občutljivosti teh tumorjev na radioterapijo in nekatere sistemske terapevtike, ki se uporabljajo za zdravljenje bolnikov s HPV-pozitivnimi tumorji ploščatoceličnega karcinoma ustnega žrela. Molekularni mehanizmi povečane občutljivosti HPV-pozitivnih celic ploščatoceličnega karcinoma ustnega žrela na radioterapijo in izbrane sistemske kemoterapevtike na osnovi platine niso popolnoma jasni, verjetno pa temeljijo na slabšem popravilu DNA. Predklinične raziskave mehanizmov delovanja radioterapije na HPV-pozitivnih tumorskih modelih so redke. Še redkejše so predklinične raziskave odziva HPV-pozitivnih tumorskih modelov ploščatoceličnega karcinoma ustnega žrela na kombinacijo obsevanja in sistemske kemoterapije s cisplatinom, ki bi omogočale boljše načrtovanje zdravljenja. Namen in hipoteze: Namen doktorske naloge je bil ugotoviti, za koliko lahko pri HPV-pozitivnih tumorjih (v primerjavi s HPV-negativnimi tumorji) zmanjšamo dozo obsevanja, ne da bi s tem zmanjšali učinek zdravljenja. Naše hipoteze so bile, da je v kombinaciji s cisplatinom radiosenzitivnost HPV-pozitivnih tumorskih celic ploščatoceličnega karcinoma ustnega žrela in vitro večja od radiosenzitivnosti HPV-negativnih tumorskih celic ploščatoceličnega karcinoma ustnega žrelada je radiosenzitivnost HPV-pozitivnih tumorskih celic večja zaradi zmanjšanega delovanja popravljalnih mehanizmov DNAter da se lahko pri HPV-pozitivnih mišjih tumorskih modelih ploščatoceličnega karcinoma ustnega žrela enak delež ozdravitve kot pri HPV-negativnih mišjih tumorskih modelih doseže z nižjo dozo obsevanja ob sočasni terapiji s cisplatinom. Metode: V raziskavah in vitro smo s pomočjo testa klonogenosti določili stopnjo občutljivosti HPV-pozitivne (2A3) in HPV-negativne (FaDu) tumorske celične linije na obsevanje, terapijo s ciplatinom in kombinacijo obeh. Ovrednotili smo razliko v deležu in časovni dinamiki popravila poškodb DNA med HPV-pozitivnimi in HPV-negativnimi tumorskimi celicami in vitro. Določili smo stopnjo eno- in dvoverižnih prelomov DNA (kometni test) in tvorbo žarišč fosforiliranih histonov &#947H2AX na DNA (ki označujejo dvoverižne prelome DNA) po obsevanju, terapiji s ciplatinom in kombinaciji obeh. Obenem smo spremljali tudi pojav posredovanega učinka sevanja (ang. bystander effect) po obsevanju HPV-pozitivnih in HPV-negativnih celic (ob sočasni terapiji s cisplatinom). Dodatno smo preverili še morfologijo celic po obsevanju v kombinaciji s cisplatinom ter ovrednotili delež apoptotičnih in mitotičnih celic. Poleg tega smo s pomočjo pretočne citometrije preverili spremembe v poteku celičnega cikla po terapiji. Isti humani tumorski celični liniji kot v poskusih in vitro smo uporabili za indukcijo tumorjev na imunsko oslabljenih laboratorijskih miših (SCID). S testom zaostanka v rasti tumorjev smo preverili odziv obeh tumorskih modelov na obsevanje ter na sočasno terapijo s cisplatinom. Določili smo tudi faktor modificiranja doze obsevanja ob sočasni terapiji s cisplatinom pri HPV-pozitivnih tumorjih za dosego enakega učinka zdravljenja kot pri HPV-negativnih tumorjih s standardnimi dozami. Rezultati: Tako v in vitro poskusih kot tudi na tumorskem modelu in vivo smo pokazali, da so HPV-pozitivne celice oziroma tumorji bolj občutljivi na samo obsevanje kot HPV-negativne celice in tumorji. Na samo terapijo z nizkimi dozami cisplatina sta bili obe tumorski celični liniji v raziskavi in vitro enako občutljivi. Tudi potenciacija učinka obsevanja z dodajanjem cisplatina je bila in vitro prisotna pri obeh tumorskih celičnih linijah. V raziskavi in vivo je bila učinkovitost dodanega cisplatina večja pri HPV-negativnih tumorjih, vendar je bil skupni učinek še vedno večji pri HPV-pozitivnih tumorjih. Faktor modificiranja doze, izračunan s primerjavo učinkovitosti obsevanja oziroma kombinirane terapije glede na HPV status tumorja, je znašal 1,3 oziroma 1,2, kar pomeni, da so HPV-pozitivni tumorji za 30 % bolje odgovorili na radioterapijo ter za 20 % bolje na kombinirano terapijo kot HPV-negativni tumorji. Ugotovili smo, da je prišlo pri HPV-pozitivnih celicah po samem obsevanju in po kombinirani terapiji do večjega deleža dvoverižnih prelomov DNA, ki so se v primerjavi s prelomi pri HPV-negativnih celicah tudi počasneje popravili. Z morfološko analizo celic smo pri kombinirani terapiji in vitro pri HPV-pozitivnih celicah opazili večji delež apoptotičnih in manjši delež mitotičnih kot pri HPV-negativnih celicah, analiza celičnega cikla pa je pri HPV-pozitivnih celicah pa terapiji pokazala zaustavitev celičnega cikla v fazi G2/M. Posredovane učinke sevanja smo opazili le pri HPV-pozitivnih celicah po kombinirani terapiji. Zaključki: V raziskavi smo potrdili, da so HPV-pozitivne celice in tumorji bolj občutljivi na obsevanje kot HPV-negativni tumorji ter da ima sočasna terapija s cisplatinom aditiven učinek ne glede na HPV status. Na podlagi rezultatov lahko predlagamo 20 % znižanje doze obsevanja pri kombinirani terapiji s cisplatinom pri HPV-pozitivnih tumorjih ploščatoceličnega karcinoma ustnega žrela, pri čemer bi dosegli enako učinkovitost kot jo dosežemo s standardno dozo pri radiokemoterapiji HPV-negativnih tumorjev. Rezultati raziskave in vitro so pokazali, da je povečana občutljivost na terapijo med drugim posledica višje ravni poškodb DNA, nižje stopnje popravila teh poškodb, zaustavitve celičnega cikla v fazi G2/M in višje stopnje apoptoze.Scientific background: Human papillomavirus (HPV) is an important etiologic factor in oropharyngeal squamous cell carcinoma. It has been established that HPV-positive oropharyngeal squamous cell carcinoma respond better to concomitant radiochemotherapy with platinum based agents than HPV-negative oropharyngeal squamous cell carcinoma. This initiated the idea of de-intensification of the treatment in patients with HPV-positive tumors. The prerequisite is to maintain the curability rate achieved with standard dose radiotherapy (fractionated irradiation with total dose of 70 Gy) and platinum-based chemotherapy, whereas the aim is to reduce the treatment-related side effects. According to the research done so far, better results of HPV-positive oropharyngeal squamous cell carcinoma treatment are the consequence of higher sensitivity of HPV-positive tumors to radio(chemo)therapy. Molecular mechanisms of the higher sensitivity of HPV-positive oropharyngeal squamous cell carcinoma to radiotherapy are not entirely clear, but are probably based on impaired DNA damage repair. Preclinical research on the effects of radiotherapy on HPV-positive tumor models is scarce and preclinical research on the response of HPV-positive oropharyngeal squamous cell carcinoma tumor models to concomitant radiochemotherapy, which could lead to better treatment planning, is even scarcer. Aim and hypotheses: The aim of this study was to determine the possible decrease in irradiation dose in HPV-positive tumors without a negative influence on the antitumor effect. Our hypotheses were that the radiosensitivity of HPV-positive oropharyngeal squamous cell carcinoma in combination with cisplatin in vitro is higher than that of HPV-negative cellsthat the radiosensitivity of HPV-positive tumor cells is higher because of impaired DNA damage repair mechanisms and lastly, that in HPV-positive tumor models the same level of cures, as in HPV-negative, can be reached with a lower irradiation dose in combination with cisplatin. Methods: In the in vitro part, we determined the response of HPV-positive and HPV-negative cell line to radiotherapy, cisplatin and the combined treatment using clonogenic assay. We evaluated the difference in the levels of DNA damage and kinetics of DNA repair in HPV-positive and HPV-negative tumor cells in vitro, using genotoxicologic studies. We evaluated the levels of single and double-strand DNA breaks (using comet assay) and the formation of foci of phosphorylated histones &#947H2AX (which are located on the sites of double-strand DNA breaks) after irradiation, after cisplatin therapy and after the combined treatment. We also monitored the presence of bystander effect after the irradiation or combined treatment in HPV-positive and HPV-negative tumor cells. Additionally, we analyzed the cell morphology after the combined treatment and evaluated the number of apoptotic and mitotic cells. We also determined the changes in the cell cycle progression after different therapies using flow cytometry. The same human tumor cell lines as in vitro were used for the induction of tumors on immunodeficient laboratory mice (SCID). We evaluated the response of both tumor models, first to radiotherapy and then to the combined treatment, using tumor growth delay assay. In the end, we determined the dose modifying factor of the irradiation dose in HPV-positive tumors after the combined treatment. Results: Both in vitro and in vivo results showed that HPV-positive cells and tumors are more sensitive to irradiation than HPV-negative cells and tumors. The treatment with low doses of cisplatin alone resulted in a comparable response in both cell lines. Potentiation of irradiation with cisplatin was also present in both cell lines. In the in vivo study, the addition of cisplatin to irradiation resulted in better potentiation in HPV-negative tumors, however, the overall response to the combined treatment was better in HPV-positive tumors. The dose modifying factor, which was evaluated by the comparison of the effectiveness of irradiation and the combined treatment according to the HPV-status of the tumor, was 1.3 for irradiation alone and 1.2 for the combined treatment. This means that HPV-positive tumors responded 30% better to radiotherapy and 20% better to the combined treatment than HPV-negative tumors. Furthermore, HPV-positive cells exhibited higher levels of double-strand DNA breaks and slower DNA damage repair after irradiation alone or in combination with cisplatin (compared to HPV-negative cells). After the combined treatment, morphologic analysis showed higher levels of apoptotic HPV-positive cells and lower levels of mitotic HPV-positive cells compared to HPV-negative cells. The analysis of the cell cycle showed arrest in G2/M phase of HPV-positive cells. Finally, bystander effect was observed only in HPV-positive cells after the combined treatment. Conclusions: The results of the study confirmed that HPV-positive cells and tumors are more sensitive to radiotherapy and that addition of cisplatin leads to an additive effect irrespective of the HPV-status. The results led to the suggestion that the irradiation dose in the concomitant treatment of HPV-positive oropharyngeal squamous cell carcinoma could be decreased for 20% in order to achieve the same level of effectiveness as in HPV-negative tumors treated with standard doses of radiochemotherapy. The results of the in vitro study showed that higher sensitivity of HPV-positive oropharyngeal squamous cell carcinoma to the described treatment is the consequence of higher levels of DNA damage, slower DNA damage repair, cell cycle arrest in G2/M phase and higher levels of apoptosis

Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity

Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity