Detection of disseminated tumor cells in aspirative drains after neck dissection.

The dissemination of individual tumor cells is a common phenomenon in solid cancers. Detection of tumor cells in bone marrow disseminated tumor cells (DTC) and in peripheral blood circulating tumor cells (CTC) in nonmetastatic situation is of high prognostic significance. Compared to breast, colon and prostate cancers, the studies on CTC and DTC in head and neck cancers are sparse. The objective of our study was to detect DTC in drains after neck dissection. Fourteen patients undergoing surgery for stages III and IV head and neck cancers were enrolled in this study--twelve presenting with squamous cell carcinoma and two with adenocarcinoma. Redon drain analysis was performed by the Cellsearch method using immunomagnetic and fluorescence approaches. A positivity threshold value was set at 2DTC/7.5 ml of the sample. Tumor cells were detected in drains of 69 % of patients a few days after surgery. The range of quantification was 3-2,094 DTC/5 ml and we showed morphological differences between the two types of carcinoma cells. DTC were detected after neck dissection both in squamous cell carcinoma and in adenocarcinoma. Potential clinical significance of tumor cells needs to be further investigated as their presence could affect pre-surgical and post-operative treatments.

Expression of pEGFR and pAKT as response-predictive biomarkers for RAS wild-type patients to anti-EGFR monoclonal antibodies in metastatic colorectal cancers.

BACKGROUND: RAS wild-type (RASw/t) tumours have been associated with better outcomes in patients with metastatic colorectal cancer (mCRC) treated with anti-EGFR monoclonal antibodies (mAb). We investigated the expression of EGFR downstream proteins under their active phosphorylated forms as potential markers in response to these patients. METHODS: One-hundred tumour samples were collected from patients with mCRC refractory to FOLFOX and/or FOLFIRI and treated by a combination of chemotherapy with anti-EGFR mAb. The outcomes were measured on response evaluation criteria in solid tumour (RECIST), progression-free survival (PFS) and overall survival (OS). All samples were assessed for RAS and BRAF mutations, and the key phosphorylated proteins of EGFR downstream signalling were quantitatively analysed using the BioPlex Protein array. RESULTS: Among the 60 RASw/t patients, 45.0% achieved a complete or partial response when treated with anti-EGFR mAb. Expression of pAKT, pERK1/2 and pMEK1 was significantly lower in RASw/t patients (P=0.0246; P=0.004; P=0.0110, respectively). The response rate was significantly higher for RASw/t patients who express pEGFR and pAKT (P=0.0258; P=0.0277, respectively). CONCLUSIONS: Overexpression of pEGFR and pAKT may predict the response rate in RASw/t patients treated with anti-EGFR mAb. On the basis of our results, we hypothesise that the association of anti-EGFR mAb and anti-AKT therapies could be of interest.

Using one-step nucleic acid amplification (OSNA) for intraoperative detection of lymph node metastasis in breast cancer patients avoids second surgery and accelerates initiation of adjuvant therapy.

BACKGROUND: Sentinel lymph node (SLN) analysis is conventionally analyzed using immunohistochemistry and in the case of SLN involvement, justifies a second surgery for axillary lymph node (ALN) resection, thus delaying the initiation of adjuvant therapies. PATIENTS AND METHODS: Three hundred and eighty-one patients with early stage breast cancer (BC) were considered in this retrospective study. SLNs were detected using combined radioisotope and dye detection. SLN involvement was analyzed using routine intraoperative One-Step Nucleic Acid Amplification (OSNA) assay, in 100 patients and compared with the conventional histopathology carried out previously in 281 patients. RESULTS: Considering positive SLNs as '++' (CK19 mRNA copy number>5000), '+' (250 < CK19 mRNA copy number <5000) and positive by inhibition in the OSNA group and macro-, micrometastases and isolated tumor cells in the histopathology group, no difference in SLN involvement rate was found between the two groups with 29.0% and 29.9% of positive SLNs, respectively. Using OSNA intraoperatively, the mean time to process the SLN was 42 min allowing immediate ALN resection, reduced significantly (P < 0.01) the re-intervention rate (9% versus 39%) and significantly (P < 0.01) accelerated the initiation of adjuvant therapy (6.2 versus 8.4 weeks). CONCLUSIONS: Using OSNA for intraoperative SLN analysis avoids second surgery for ALN resection in most patients and accelerates initiation of adjuvant therapy.

Intradermal tacrolimus prevent scar hypertrophy in a rabbit ear model: a clinical, histological and spectroscopical analysis.

BACKGROUND: Keloids and hypertrophic scars (HSc) affect 4.5-16% of the population. Thus far, the different approaches of keloid treatment are not very efficient, with a 50% relapse rate and many ongoing researches are looking for simple, safe and more efficient therapeutic methods. Tacrolimus is an immunomodulator that could be useful in treating keloid. OBJECTIVES: The objective of this study is to evaluate the effectiveness of Tacrolimus in inhibiting HSc formation on rabbits' ears model and to check optical skin spectroscopy in tissue characterization. METHODS: Our study was carried out on 20 New-Zealand female white rabbits. HSc were obtained by wounding rabbits' ear. These wounds were treated with intradermal injections of tacrolimus (0.2-0.5 mg/cm(2)) or a vehicule. The assessment of treatment efficacy was performed by clinical examinations, histological assay and skin spectrometry. RESULTS: Tacrolimus did not induce general or local side-effects. The scar elevation index in treated subjects was half less than that of the untreated ones. Furthermore, dermal thickness and inflammatory cellular density were both significantly smaller for treated scars than for the control ones. In vivo optical skin spectroscopy can characterize hypertrophic and normal skin with high sensibility and specificity. CONCLUSION: Intradermal injection of tacrolimus at 0.5 mg/cm(2) is an efficient way to prevent HSc in our experiment model and its tolerance is correct. Optical spectroscopy could be a good non-invasive tool to evaluate HSc treatment. These promising results might be proposed for patients suffering from keloid.

Comparison of Three Real-Time PCR Assays for the Detection of PIK3CA Somatic Mutations in Formalin-Fixed Paraffin Embedded Tissues of Patients with Breast Carcinomas.

Breast cancer is the leading cause of cancer-related death in women worldwide. Mutations of the PIK3CA gene are found in approximately 25% of breast carcinomas and are reported as activators of the PI3K/AKT/mTOR pathway. This study aims to compare three assays for the somatic mutation detection of PIK3CA gene in FFPE tissues of patients with breast cancer. We compared Cobas® PIK3CA Mutation Test (Roche Diagnostics, Meylan, France), PCR amplification-refractory mutation system Scorpions® (ARMS) and High-Resolution Melting PCR assay (HRM) for the detection of PIK3CA mutations. Discrepant samples were assessed using Next Generation Sequencing (NGS). 46 FFPE breast carcinomas samples of patients treated for breast cancer have been assessed for PIK3CA mutations using the three PCR assays. Among the 46 samples, 17 (37.8%), 13 (28.36%) and 19 (41.3%) had a PIK3CA mutation, with Cobas®, ARMS and HRM assays respectively. Three different mutations of PIK3CA have been detected for one sample. Calculated kappa were 0.95[0.86;1] between Cobas® and HRM, 0.75[0.55;0.95] between Cobas® and ARMS and 0.72[0.51;0.92] between HRM and ARMS. Five samples were found with discrepant results. Our study shows that the Cobas® assay is suitable for PIK3CA mutation assessment in patients with breast cancer. HRM assay is also suitable for PIK3CA mutation assessment but requires a mutation characterization with a specific assay.

RILA blood biomarker as a predictor of radiation-induced sarcoma in a matched cohort study.

PURPOSE: Radiation-induced sarcoma (RIS) is a rare but serious event. Its occurrence has been discussed during the implementation of new radiation techniques and justified appropriate radioprotection requirements. New approaches targeting intrinsic radio-sensitivity have been described, such as radiation-induced CD8 T-lymphocyte apoptosis (RILA) able to predict late radio-induced toxicities. We studied the role of RILA as a predisposing factor for RIS as a late adverse event following radiation therapy (RT). PATIENTS AND METHODS: In this prospective biological study, a total of 120 patients diagnosed with RIS were matched with 240 control patients with cancer other than sarcoma, for age, sex, primary tumor location and delay after radiation. RILA was prospectively assessed from blood samples using flow cytometry. RESULTS: Three hundred and forty-seven patients were analyzed (118 RIS patients and 229 matched control patients). A majority (74%) were initially treated by RT for breast cancer. The mean RT dose was comparable with a similar mean (± standard deviation) for RIS (53.7 ±â€¯16.0 Gy) and control patients (57.1 ±â€¯15.1 Gy) (p = .053). Median RILA values were significantly lower in RIS than in control patients with respectively 18.5% [5.5-55.7] and 22.3% [3.8-52.2] (p = .0008). Thus, patients with a RILA >21.3% are less likely to develop RIS (p < .0001, OR: 0.358, 95%CI [0.221-0.599]. CONCLUSION: RILA is a promising indicator to predict an individual risk of developing RIS. Our results should be followed up and compared with molecular and genomic testing in order to better identify patients at risk. A dedicated strategy could be developed to define and inform high-risk patients who require a specific approach for primary tumor treatment and long term follow-up.

[Molecular diagnosis and response prediction to anti-tyrosine kinase receptors in oncology]. / Diagnostic moléculaire et prédiction de réponse aux anti-récepteurs tyrosine kinase en cancérologie.

Tyrosine kinase receptors are an important family of membrane receptors implicated in oncogenesis. Their activation triggers signaling cascades that activate cell growth, proliferation and controls cell death. Inhibiting these receptors leads to signaling impairments and favors antitumor activity. Different anti-receptor tyrosine kinases are proposed for therapy, mainly consisting in monoclonal antibodies impairing the fixation of ligands onto the receptors, and of tyrosine kinase inhibitors, blocking the phosphorylation of the receptors. These therapeutic agents are being extensively studied and supported by considerable scientific and financial investments in the pharmaceutical industry. In this paper, we will consider Human Epidermal Growth Factor Receptors (HERs). Therapeutic use of targeted therapy agents, directed against these receptors is conditioned by the detection of target receptor expression in the tumor tissues. However, it appears more and more clearly that additional diagnosis molecular markers as well as surrogate response prediction markers are needed to enable more efficacious therapeutics.

Correlation between radio-induced lymphocyte apoptosis measurements obtained from two French centres.

PURPOSE OF THE RESEARCH: In the era of modern treatment delivery, increasing the dose delivered to the target to improve local control might be modulated by the patient's intrinsic radio-sensitivity. A predictive assay based on radio-induced lymphocyte apoptosis quantification highlighted the significant correlation between CD4 and CD8 T-lymphocyte apoptosis and grade 2 or 3 radiation-induced late toxicities. By conducting this assay at several technical platforms, the aim of this study was to demonstrate that radio-induced lymphocyte apoptosis values obtained from two different platforms were comparable. MATERIALS AND METHODS: For 25 patients included in the PARATOXOR trial running in Dijon the radio-induced lymphocyte apoptosis results obtained from the laboratory of Montpellier (IRCM, Inserm U1194, France), considered as the reference (referred to as Lab 1), were compared with those from the laboratory located at the Institut de cancérologie de Lorraine (ICL, France), referred to as Lab 2. Different statistical methods were used to measure the agreement between the radio-induced lymphocyte apoptosis data from the two laboratories (quantitative data). The Bland-Altman plot was used to identify potential bias. RESULTS: All statistical tests demonstrated good agreement between radio-induced lymphocyte apoptosis values obtained from both sites and no major bias was identified. CONCLUSIONS: Since radio-induced lymphocyte apoptosis values, which predict tolerance to radiotherapy, could be assessed by two laboratories and showed a high level of robustness and consistency, we can suggest that this assay be extended to any laboratories that use the same technique.

[Sarcoma developed in irradiated area: Preliminary results of the SARI trial]. / Sarcomes développés en territoire irradié : résultats préliminaires de l'étude SARI.

Radiotherapy and surgery are the two main pillars of the locoregional treatment of cancer. The risk of second malignancy is better evaluated and constitutes a major issue regarding radioprotection of the patients. Among malignant disease observed in the surviving irradiated patients, the occurrence of sarcoma is a rare event but associated with a poor outcome since the 5 year overall survival is estimated at 10 to 35 %. The SARI protocol, written in 2011, included 120 patients and 240 controlled patients, irradiated in the same conditions but without sarcoma observed during the follow up. The main objective was to identify the clinical and biological factors associated with the occurrence of such a complication. The secondary objective was to identify the dosimetric characteristics of the treatment of the primary. Preliminary results will be presented during the 2016 meeting of the French radiation oncology society.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

In vivo potentiation of radiation response by topotecan in human rhabdomyosarcoma xenografted into nude mice.

The lack of new highly efficacious drugs for cancer treatment promotes the search for innovative therapeutic modalities. The authors reported the results leading to the definition of parameters needed to demonstrate a possible radiopotentiation by topotecan (TPT) on two representative human rhabdomyosarcomas (RMSs) xenografted into nude mice. Experimental studies of radiopotentiation with different doses of topotecan showed that concomitant association of topotecan and RT for 5 consecutive days provided a synergistic therapeutic effect. Response rates were statistically higher with the radiochemotherapeutic combination (P < 0.001). Efficacy enhancement factors of this combination compared with the sum of the antitumoral activity of these treatments separately administrated were 1.54 and 1.60, respectively, on both rhabdomyosarcomas. Moreover, the efficiency of the combination of radiotherapy at the dose of 20 Gy with topotecan (12.5 mg/kg) was not statistically different from that of radiotherapy at the dose of 40 Gy. According to microscopy results, the analyses performed at different periods after topotecan treatment alone, radiotherapy alone, and their combination seemed to show that tumoral repopulation by malignant cells is as fast as the dose of radiotherapy and/or topotecan is low. Furthermore, lesions observed with the dose of 40 Gy were similar to those obtained with the association of topotecan at the dose of 12.5 mg/kg and radiotherapy at the dose of 20 Gy. In conclusion, all clinical and pathological results are consistent with a radiopotentiation effect of topotecan on the two xenografted human rhabdomyosarcomas and are currently leading to the design of clinical studies.

Clinical impact of pharmacokinetically-guided dose adaptation of 5-fluorouracil: results from a multicentric randomized trial in patients with locally advanced head and neck carcinomas.

A significant link between 5-fluorouracil (5FU) plasma concentration and its therapeutic activity has been demonstrated in colon and head and neck cancer patients for 5FU used as a continuous infusion. Dose adjustment based on pharmacokinetic follow-up has been proposed to decrease hematological and digestive toxicities, but the clinical impact of this approach has not yet been demonstrated. A randomized multicentric study was conducted to evaluate the clinical interest of 5FU dose adaptation guided by pharmacokinetics. One hundred twenty-two head and neck cancer patients were randomly assigned to receive induction chemotherapy with cisplatin (100 mg/m2, day 1) and 5FU (96-h continuous infusion), either at standard dose (St-arm; 4 g/m2) or at a dose adjusted according to the 5FU area under the curve (AUC0-48h; PK-arm). In total, 106 patients were evaluable for toxicity and response. In the PK-arm (n = 49), 5FU doses and area under the curve were significantly reduced during cycle 2 and cycle 3 (P < 0.001) as compared with the St-arm (n = 57). Grade 3-4 neutropenia and thrombopenia were significantly more frequent in the St-arm as compared with the PK-arm (17.5% versus 7.6%, respectively; P = 0.013). No grade 3-4 mucositis occurred in the PK-arm, whereas 5.1% was observed in the St-arm (P < 0.01). The objective response rate was comparable in the two treatment arms: 77.2% in the St-arm versus 81.7% in the PK-arm. The present study is the first to demonstrate, in a randomized design, the clinical interest of an individual 5FU dose adaptation based on pharmacokinetic survey, in terms of therapeutic index improvement.

Human growth hormone gene transfer into tumor cells may improve cancer chemotherapy.

Chemotherapy remains the main tool for the treatment of cancers, but is often hampered by tumor cell resistance. In this context, the transfer of genes able to accentuate the effect of anticancer drugs may constitute a useful approach, as exemplified by inactivation of nuclear factor (NF)-kappa B via direct transfer of a gene encoding a negative dominant of its natural inhibitor I kappa B, leading to improved response to cancer chemotherapy. Following our previous report that transfection of human growth hormone (hGH) gene into human monocytic cell lines may also inactivate NF-kappa B in another situation, we decided to test the consequences of hGH gene transfer on cancer treatments. We demonstrated that hGH-transfected human myeloid leukemia U937 cells were sensitized to an apoptotic signal mediated by the anticancer drugs. In parallel, we found that, by inhibiting degradation of I kappa B, hGH gene transfer diminished NF-kappa B entry into the nuclei of U937 cells exposed to daunorubicin. Finally, we report that hGH-transfected tumor cells engrafted in nude mice responded in vivo to chemotherapy with nontoxic doses of daunorubicin whereas, under the same conditions, control tumor cells remained insensitive. Overall, this study therefore suggests that hGH gene transfer may offer new therapeutic prospects in cancer therapy.

Improvement of nonviral p53 gene transfer in human carcinoma cells using glucosylated polyethylenimine derivatives.

Polyethylenimine (PEI) derivatives are potent polycationic nonviral vectors for gene transfer. The gene transfer efficiency of glucosylated and galactosylated PEI derivatives was optimized using green fluorescent protein gene as reporter gene in FaDu and PANC3 human carcinoma cell lines. Glucosylated or galactosylated PEI derivatives were found to be slightly less cytotoxic than unsubstituted PEI. Gene transfer efficiency was found to be related to DNA/cell number ratio and optimal gene transfer efficiency was achieved at 4 microg DNA/10(5) cells. PEI-DNA complexes were found to enter cells rapidly and were detected into cytoplasmic vesicles 2 hours post-transfection. Green fluorescent protein gene expression was detected 4-6 hours after transfection and reached maximal value 24 hours post-transfection. The results achieved demonstrated that glucosylated PEI yield higher and longer gene transfer efficiency than unsubstituted PEI. Using glucosylated PEI allowed to achieve significant gene transfer in more than 10% of the total cell population for more than 4 days. These data were then applied to p53 gene transfer in PANC3 cells bearing p53 gene deletion and consequently unable to initiate apoptosis. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of p53 mRNA expression and transient P53 protein expression. P53 protein functionality was further demonstrated because transfected cells underwent apoptosis.

Encapsulation of doxorubicin in thermosensitive small unilamellar vesicle liposomes.

The optimisation of the formulation of thermosensitive, doxorubicin-containing small unilamellar liposomes is described. The liposomes were first strictly defined in terms of size distribution and size stability and a quality level was defined. The suspension contained more than 95% vesicles with a maximal diameter of 50 nm and kept this level for a minimum of 24 hours. Several lipid mixtures were tested in defined thermal conditions usable for in vitro experiments: 43 degrees C in fetal calf serum-containing medium. The mixture yielding the best differential thermal stability (DTS) defined as the difference of release between 37 degrees C and 43 degrees C exposures was found to be a dipalmitoylphosphatidylcholine/distearoylphosphatidyl-choline/chol esterol mixture in 5:4:2 molar ratio yielding 72% DTS. These thermosensitive liposomes were evaluated between pH 6.00 and 8.00 since hyperthermia-induced lethality was reported to be enhanced by pH variations. Their release capacity was not altered by any pH variations. Incorporation of doxorubicin within these liposomes was then performed. The release kinetics at 37 degrees and 43 degrees C were determined. It is proposed to use this formulation in in vitro experiments on tumour cells, although a decrease of DTS was evident.

In vitro evaluation of the association of thermosensitive liposome-encapsulated doxorubicin with hyperthermia.

Doxorubicin was encapsulated in small unilamellar thermosensitive liposomes which were strictly defined in terms of size distribution and size stability: more than 95% of vesicles with a maximal diameter of 50 nm stable for a minimum of 24 hours. In addition, the preparation procedure was optimised to achieve the highest differential thermal stability defined as the difference of release between 37 degrees and 43 degrees C exposures in serum-containing medium (dipalmitoylphosphatidylocholine/distearoylphosphatidylcholine/cho lesterol mixture in 5:4:2 molar ratio). The cytotoxicity of thermosensitive-liposome encapsulated doxorubicin was then evaluated in combination with 43 degrees C hyperthermia on HelaS3 human tumour cells using colony-forming assays. Results confirmed that hyperthermia potentiates the cytotoxic effects of doxorubicin. Liposome encapsulation was found to further enhance these effects when 0.05 mumol/l doxorubicin concentration was used.

Antiproliferative activity of thermosensitive liposome-encapsulated doxorubicin combined with 43 degrees C hyperthermia in sensitive and multidrug-resistant MCF-7 cells.

Thermosensitive liposome-encapsulated doxorubicin (TLED) was compared to free doxorubicin, at 37 degrees C or combined with 43 degrees C hyperthermia, in sensitive and multidrug-resistant MCF-7 human tumour cells using clonogenic assays. In the resistant subline, TLED was found to partly circumvent multidrug resistance (MDR). The reversal was comparable to that obtained when verapamil was added to free doxorubicin. When hyperthermic treatment was applied, no difference in thermosensitivity was found between sensitive and resistant cells. The combination of hyperthermia with free doxorubicin did not reverse MDR. Hyperthermia and TLED yielded additive effects in the resistant cells while potentiation was observed in the sensitive cells. These results confirmed the usefulness of the liposome encapsulation of doxorubicin in reversing MDR. The possibility of obtaining additive cytotoxicity using TLED combined with hyperthermia may represent an alternative way of intensification of doxorubicin cytotoxicity concomitant with the circumvention of MDR without using MDR reversing agents, which often generate limiting toxic side-effects.

MTT assays allow quick and reliable measurement of the response of human tumour cells to photodynamic therapy.

MCF-7 and HT-29 cell lines were selected as a reliable model to examine the possible parameters affecting the sensitivity of tumour cells to photodynamic therapy (PDT) using a dye-laser at 630 nm. The chemical composition of haematoporphyrin derivative (HPD) was determined by high-performance liquid chromatography (HPLC) analysis and was in agreement with reported values. MTT assays were performed to assess the time-dependency of PDT and the influence of the output power and light fluence. The results showed a maximal cytotoxicity 48 h after photoirradiation. The output power (1 or 2 W) did not significantly affect the cytotoxicity when the fluence was constant (20 J/cm2). However, an increase in fluence (10-40 J/cm2) led to a significant enhancement of cytotoxicity until maximal values were reached (30-40 J/cm2). A further increase in fluence (50 J/cm2) proved to induce a fall-off in cytotoxicity related to the intense photobleaching of HPD.

Radiosensitivity of multicellular tumour spheroids obtained from human ovarian cancers.

The radioresponsiveness of immunologically characterised (KL1, antivimentin and OC125) human ovarian carcinoma cells, obtained from effusions or solid tumours, was assayed in vitro using the multicellular tumour spheroids (MTS) three-dimensional model. Great interspecimen variabilities were observed in MTS doubling time (1.0-8.5 days), as well as in the doses inducing a 50% decrease in the MTS individual volume (ID50) (0.56-9.15 Gy), or in the overall population MTS number (SCD50) (1.9-15.7 Gy) and the residual/initial MTS individual volume ratio after 2 Gy irradiation (RSV2) (10-88%). The doubling time, DNA-ploidy and S-phase fraction did not correlate with the ID50. Significant correlations were found between the new parameters defined (RSV2 and ID50) and the SCD50, a well-accepted local control parameter. These parameters demonstrated their usefulness for studying the radiosensitivity of MTS prepared from human ovarian tumour biopsies.

Constitutive NF-kappaB activity influences basal apoptosis and radiosensitivity of head-and-neck carcinoma cell lines.

PURPOSE: Nuclear factor-kappaB (NF-kappaB) has been implicated in anti-apoptotic gene transactivation, according to its transcriptional activity. The present study was designed to investigate whether constitutive NF-kappaB activity could modulate basal apoptosis and intrinsic radiosensitivity of KB head-and-neck carcinoma cell line and KB3 subline. The KB3 subline was more radiosensitive (SF2 = 0.48, alpha = 0.064) than the radioresistant KB parental cell line (SF2 = 0.80, alpha = 0.114). METHODS AND MATERIALS: Constitutive NF-kappaB DNA-binding activity was determined using electrophoretic mobility shift assay. Modulation of NF-kappaB activity was performed by exposing both cell lines to tumor necrosis factor alpha or dexamethasone. Apoptotic cell population was analyzed using flow cytometry (annexin V/propidium iodide). Radiosensitivity was assessed from determination of the surviving fraction at 2 Gy (SF2), and alpha and beta parameters were determined using the linear-quadratic model. RESULTS: Constitutive NF-kappaB activity was found to be significantly lower in KB3 than in KB. KB cell line exposure to dexamethasone significantly decreased NF-kappaB DNA-binding activity and, consequently, enhanced baseline apoptosis and radiosensitivity (alpha values: 0.114 vs. 0.052). Conversely, exposure of KB3 cells to tumor necrosis factor alpha increased NF-kappaB DNA-binding activity and resulted in a significant decrease (50%) in rate of apoptosis and in radiosensitivity (SF2 values: 0.48 vs. 0.63). CONCLUSIONS: Modulation of NF-kappaB DNA-binding activity influences baseline apoptosis and intrinsic radiosensitivity.