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 emerging; however, 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.

Background 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 assay; post irradiation cell cycle distribution was studied by flow cytometry; the 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 LDHRS-positive 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.

Synergistic effect of cisplatin chemotherapy combined with fractionated radiotherapy regimen in HPV-positive and HPV-negative experimental pharyngeal squamous cell carcinoma.

HPV infection renders oropharyngeal squamous cell carcinomas more radiosensitive, which results in a favorable prognosis for HPV-positive patients treated with radiation alone or with concurrent platinum-based chemotherapy. The degree of radiosensitivity in fractionated regimens has not yet been fully explored; therefore, in this study, the radiosensitivity of HPV-negative tumors (FaDu) was compared to that of HPV-positive tumors (2A3) subjected to concurrent cisplatin chemotherapy and fractionated versus isoeffective single-dose tumor irradiation in immunodeficient mice. HPV-positive tumors were approximately 5 times more radiosensitive than HPV-negative tumors, irrespective of the irradiation regimen. In both tumor models, concurrent cisplatin chemotherapy and the fractionated regimen induced significant tumor radiosensitization, with a 3- to 4-fold increase in the tumor growth delay compared to that of single-dose irradiation. Furthermore, the degree of radiosensitization induced by cisplatin chemotherapy concurrent with the fractionated irradiation regimen was much higher in HPV-positive tumors, where a synergistic antitumor effect was observed. Specifically, after combined therapy, a 26% higher survival rate was observed in mice with HPV-positive tumors than in mice with HPV-negative tumors. These data suggest that HPV-positive tumors are more radiosensitive to fractionated regimen than to single-dose irradiation with concurrent cisplatin chemotherapy acting synergistically to irradiation.

Mechanisms of different response to ionizing irradiation in isogenic head and neck cancer cell lines.

BACKGROUND: Treatment options for recurrent head and neck tumours in the previously irradiated area are limited, including re-irradiation due to radioresistance of the recurrent tumour and previous dose received by surrounding normal tissues. As an in vitro model to study radioresistance mechanisms, isogenic cells with different radiosensitivity can be used. However, they are not readily available. Therefore, our objective was to establish and characterize radioresistant isogenic human pharyngeal squamous carcinoma cells and to evaluate early radiation response in isogenic parental, radioresistant and radiosensitive cells. METHODS: Radioresistant cells were derived from parental FaDu cells by repeated exposure to ionizing radiation. Radiosensitivity of the established isogenic radioresistant FaDu-RR cells was evaluated by clonogenic assay and compared to isogenic parental FaDu and radiosensitive 2A3 cells. Additional phenotypic characterization of these isogenic cells with different radiosensitivity included evaluation of chemosensitivity, cell proliferation, cell cycle, radiation-induced apoptosis, resolution of DNA double-strand breaks, and DNA damage and repair signalling gene expression before and after irradiation. RESULTS: In the newly established radioresistant cells in response to 5 Gy irradiation, we observed no alteration in cell cycle regulation, but delayed induction and enhanced resolution of DNA double-strand breaks, lower induction of apoptosis, and pronounced over-expression of DNA damage signalling genes in comparison to parental cells. On the other hand, radiosensitive 2A3 cells were arrested in G2/M-phase in response to 5 Gy irradiation, had a prominent accumulation of and slower resolution of DNA double-strand breaks, and no change in DNA damage signalling genes expression. CONCLUSIONS: We concluded that the emergence of the radioresistance in the established radioresistant isogenic cells can be at least partially attributed to the enhanced DNA double-strand break repair, altered expression of DNA damage signalling and repair genes. On the other hand, in radiosensitive isogenic cells the reduced ability to repair a high number of induced DNA double-strand breaks and no transcriptional response in DNA damage signalling genes indicate on a lack of adaptive response to irradiation. Altogether, our results confirmed that these isogenic cells with different radiosensitivity are an appropriate model to study the mechanisms of radioresistance.

Electrochemotherapy of radioresistant head and neck squamous cell carcinoma cells and tumor xenografts.

Electrochemotherapy is an established local ablative method used for the treatment of different tumor types, including tumors of the head and neck area. Clinical studies have demonstrated a lower response rate of tumors that recur in pre­irradiated area. The aim of the present study was to explore the response of experimentally induced radioresistant cells and tumors to electrochemotherapy with cisplatin or bleomycin. The radioresistant cells (FaDu­RR) were established by fractionated irradiation of parental human squamous cell carcinoma cell line, FaDu. We compared the 2 cell lines in response to chemotherapy and electrochemotherapy with cisplatin or bleomycin in vitro and in vivo. Using specific mass spectrometry­based analytical methods we determined the difference in the uptake of chemotherapeutics in tumors after electrochemotherapy. Additionally, we compared the capacity of the cells to repair DNA double­strand breaks (DSB) after exposure to the drugs used in electrochemotherapy with the γH2AX foci resolution determined by immunofluorescence microscopy. Our results indicate radio­ and cisplatin cross­resistance, confirmed with the lower response rate of radioresistant tumors after electrochemotherapy with cisplatin. On the other hand, the sensitivity to electrochemotherapy with bleomycin was similar in both cell lines and tumors. While the uptake of chemotherapeutics after electrochemotherapy was comparable in both tumor models, there was a difference between the cell lines in capacity to repair DNA DSB­the radioresistant cells had a lower level of DSB and faster DNA repair rate after exposure to both, cisplatin or bleomycin. Due to the higher complete response rate after electrochemotherapy with bleomycin than with cisplatin, we conclude that the results favor bleomycin­over cisplatin­based electrochemotherapy for treatment of radioresistant tumors and/or tumors that regrow after radiotherapy.

Intravital Monitoring of Vasculature After Targeted Gene Therapy Alone or Combined With Tumor Irradiation.

Vascular-targeted therapies exhibit radiosensitizing effects by remodeling tumor vasculature, thus facilitating the increased oxygenation of the remaining tumor tissue. To examine these phenomena, the effects of antiendoglin gene therapy alone and in combination with irradiation were monitored for 5 consecutive days on a murine mammary adenocarcinoma (TS/A) tumor model growing in a dorsal window chamber. The vascularization of the tumors was assessed by the determination of the tumor vascular area and by measurement of tumor perfusion by using laser Doppler flowmetry to provide insight into intratumoral gene electrotransfer effects. The changes in the vascular area after this specific therapy correlated with laser Doppler measurements, indicating that either of the methods can be used to demonstrate the induced changes in the vascularization and perfusion of tumors. Gene electrotransfer with an endothelial-specific promoter resulted in a vascular-targeted effect on tumor vasculature within the first 24 hours and did not restore within 5 days. The combination with the irradiation did not result in a more pronounced vascular effect, and irradiation alone only abrogated the formation of new vessels and prevented an increase in the tumor perfusion over time. The results indicate that tumors grown in a dorsal window chamber facilitate intravital measurements of the vascularization of tumors and blood perfusion, enabling the monitoring of the antiangiogenic or vascular disruptive effects of different therapies.

Electrochemotherapy with cisplatin or bleomycin in head and neck squamous cell carcinoma: Improved effectiveness of cisplatin in HPV-positive tumors.

Human papillomavirus (HPV) is an important etiological factor in head and neck squamous cell carcinomas (SCCs). Standard treatment of HPV-positive tumors with platinum-based radio(chemo)therapy results in a better outcome than in HPV-negative tumors. Electrochemotherapy is becoming an increasingly recognized mode of treatment in different cancers; thus, its use in the management of head and neck SCC is of considerable interest. However, response to electrochemotherapy according to HPV status of the tumors has not been evaluated yet. Thus, our aim was to compare the effect of electrochemotherapy with cisplatin or bleomycin between HPV-negative and HPV-positive human pharyngeal SCC derived cell lines and tumor models. HPV-positive cells and tumors were found to be more sensitive to electrochemotherapy with cisplatin than HPV-negative ones, whereas sensitivity to electrochemotherapy with bleomycin was similar irrespective of the HPV status. The higher sensitivity of HPV-positive cells and tumors to electrochemotherapy with cisplatin is likely due to the higher level and slower repair of DNA damage. In HPV-negative tumors, a higher number of complete responses was recorded after bleomycin-based rather than cisplatin-based electrochemotherapy, while in HPV-positive tumors electrochemotherapy with cisplatin was more effective.

Dose-Modifying Factor of Radiation Therapy with Concurrent Cisplatin Treatment in HPV-Positive Squamous Cell Carcinoma: A Preclinical Study.

Human papillomavirus (HPV) is an important etiological factor in oropharyngeal squamous cell carcinoma (SCC). Compared to HPV-negative tumors, HPV-positive oropharyngeal SCC has shown a better response to nonsurgical treatments. In this study, we determined the dose-modifying factors for HPV-positive tumors with single-dose irradiation, with or without low radiosensitizing doses of cisplatin. In vitro, we determined an increased radiosensitivity of HPV-positive SCC, which might be a consequence of HPV-induced changes in the cell cycle regulation and DNA damage response, leading to increased cell death. Additionally, compared to HPV-negative tumors, 30% higher radiosensitivity of HPV-positive tumors was determined by tumor growth delay monitoring in immunodeficient mice in vivo. Concurrent cisplatin treatment had an additive effect in both HPV-negative and HPV-positive tumors, resulting in 20% better response in HPV-positive tumors than in HPV-negative tumors.

Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy.

Pre-clinical and clinical data indicate differences in the responses of melanoma and carcinoma tumours to electrochemotherapy. The purpose of this study was to investigate the origin of this difference, whether it is due to the intrinsic difference in tumour cell susceptibility to the chemotherapeutic, or due to the tumour micro-environment. For this purpose, we performed a pre-clinical study in B16F1 melanoma and TS/A carcinoma tumours in mice, in which the antitumour effectiveness of electrochemotherapy with bleomycin, the intrinsic sensitivity of tumour cells in vitro, the pharmacokinetics of bleomycin in plasma and tumours, and the vascularization of tumours in vivo were evaluated. The results of the treatment show that carcinoma was significantly more responsive to electrochemotherapy than melanoma. This effect cannot be ascribed to the intrinsic sensitivity of these cells, as melanoma cells were more sensitive than carcinoma cells in vitro. The difference in responses could be ascribed to differences in the pharmacokinetics of bleomycin; at the time of electroporation in carcinomas, more bleomycin was accumulated. This effect could be due to differences in tumour vascularization, as carcinoma tumours had numerous well-distributed, small blood vessels, while melanomas were less vascularized, exhibiting predominantly larger vessels. In conclusion, this study provides evidence on the importance of the tumour micro-environment, particularly the tumour vasculature, in the responses of the tumours to bleomycin electrochemotherapy. Vasculature is important for the pharmacokinetics of bleomycin, influencing drug accumulation and drug distribution in tumours, and might be used as a predictive factor for the tumour response to electrochemotherapy.

Bystander Effect Induced by Electroporation is Possibly Mediated by Microvesicles and Dependent on Pulse Amplitude, Repetition Frequency and Cell Type.

Bystander effect, a known phenomenon in radiation biology, where irradiated cells release signals which cause damage to nearby, unirradiated cells, has not been explored in electroporated cells yet. Therefore, our aim was to determine whether bystander effect is present in electroporated melanoma cells in vitro, by determining viability of non-electroporated cells exposed to medium from electroporated cells and by the release of microvesicles as potential indicators of the bystander effect. Here, we demonstrated that electroporation of cells induces bystander effect: Cells exposed to electric pulses mediated their damage to the non-electroporated cells, thus decreasing cell viability. We have shown that shedding microvesicles may be one of the ways used by the cells to mediate the death signals to the neighboring cells. The murine melanoma B16F1 cell line was found to be more electrosensitive and thus more prone to bystander effect than the canine melanoma CMeC-1 cell line. In B16F1 cell line, bystander effect was present above the level of electropermeabilization of the cells, with the threshold at 800 V/cm. Furthermore, with increasing electric field intensities and the number of pulses, the bystander effect also increased. In conclusion, electroporation can induce bystander effect which may be mediated by microvesicles, and depends on pulse amplitude, repetition frequency and cell type.