p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines.

The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity. In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants alpha and beta in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4-10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4 +/- 0.1 to 2.6 +/- 0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly (P < 0.01) more radioresistant (mean inactivation dose 2.23 +/- 0.15 Gy) than cell lines which lacked a wild-type gene (1.82 +/- 0.24 Gy). Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.

Effects of paclitaxel with or without cremophor EL on cellular clonogenic survival and apoptosis.

Paclitaxel is currently formulated in a vehicle of 50% ethanol and 50% polyethoxylated surfactant cremophor EL. Cremophor EL has been reported to reverse P-glycoprotein-mediated multidrug resistance (MDR) at doses which are clinically achievable. It has also been reported to have a cytotoxic effect per se. In this study we used two different methods to evaluate the survival of cells exposed to paclitaxel with or without cremophor EL and the vehicle alone. Two laryngeal SCC cell lines (UT-SCC-19A and UT-SCC-29) and two ovarian adenocarcinoma cell lines (UT-OC-3 and UT-OC-5) established in our laboratory were investigated. Northern hybridisation was used to study the mdr-1 mRNA expression of the cell lines. With sensitive Northern analyses, these four lines yielded mdr-1 mRNA signals of the expected 4.5 kb size and of variable intensity, generally at higher levels than those in the positive control cell line KB. The 96-well plate clonogenic assay was used to obtain the fraction survival data and apoptosis was recorded by time-lapse video microscopy. Both methods indicate that cremophor EL alone has no effect on cellular survival. Consequently, paclitaxel without cremophor EL is as active as paclitaxel with cremophor EL in vitro.

Paclitaxel-induced apoptotic changes followed by time-lapse video microscopy in cell lines established from head and neck cancer.

Paclitaxel (Taxol) is a potent chemotherapeutic drug for squamous-cell carcinoma (SCC) of the head and neck in vitro with microtubule-stabilizing activity that arrests cells in G2-M. To study the mechanism of its cytotoxic effect on SCC in vitro, we exposed five laryngeal SCC cell lines to 10 nM paclitaxel. The cell lines were studies by time-lapse video microscopy for 96 h, and by agarose gel electrophoresis. Paclitaxel blocked the cells in the premitotic phase for 6-24 h, after which the cells died morphologically by apoptosis. Mitotically arrested cells were seen within a few minutes after exposure to paclitaxel. No mitoses were seen in the paclitaxel-treated cells. A few apoptoses were also seen in the control cultures grown without paclitaxel, but they represented only 6%-20% of the frequency of apoptoses seen in the paclitaxel-treated group. In some paclitaxel-treated cultures the cells escaped the mitotic arrest without cytokinesis and formed multinucleated cells that eventually died. Agarose gel electrophoresis showed oligonucleosomal DNA fragmentation characteristic of apoptosis. We conclude that time-lapse video microscopy is an efficient method of observing drug-induced morphological changes in cell culture. Paclitaxel at a 10 nM concentration rapidly induces a premitotic block, which usually leads to apoptotic cell death. In some cases multinucleated cells are formed that morphologically also eventually die by apoptosis.

Paclitaxel and irradiation induce apoptosis in squamous cell carcinoma cell lines in an additive way.

Paclitaxel (Taxol) is a new antimicrotubule plant product, which not only stabilizes the microtubules by inhibiting their disassembly but also promotes the assembly of microtubules. This causes a block in the G2-M phase of the cell cycle known to be a radiosensitive phase of the cycle. Previously we demonstrated that 10 nM paclitaxel accumulated the cells of laryngeal carcinoma cell lines in the G2-M phase as measured by flow cytometry. Time-lapse videomicroscopy demonstrated that the cells died morphologically by apoptosis after a premitotic block. Agarose gel electrophoresis showed the DNA-laddering typical of apoptosis. To investigate the effects of irradiation and paclitaxel separately and concomitantly, we used five newly established laryngeal carcinoma cell lines. The effects were recorded with time-lapse videomicroscopy over 96 hours on controls, cells irradiated with 2 Gy, cells exposed to 1 nM or 5 nM paclitaxel and cells irradiated with 2 Gy after incubating in 1 nM or 5 nM paclitaxel for 24 hours. Spontaneous apoptoses were seen in all cell lines tested. The 2 Gy irradiation dose induced a propagated apoptotic response in two of these cell lines. In all cell lines paclitaxel induced a premitotic block only in some cells at the tested concentrations and these cells died morphologically by apoptosis, whereas irradiation and paclitaxel concomitantly caused an additive inhibition in mitotic activity and caused an additive apoptotic response. An additive effect of paclitaxel and radiation was seen with doses readily achievable in clinical treatment. This additive effect seems to be due to other mechanisms of action than the premitotic block.

The effect of irradiation on mitotic and apoptotic frequency in head and neck cancer cell lines, the correlation to p53 mutations and clonogenic survival.

It was our intention to enlighten the controversy between the mainstream of studies and our previous results showing a correlation between the intrinsic radiosensitivity and the p53 allele status of 20 human head and neck squamous cell carcinoma (HNSCC) cell lines. In our study cell lines carrying a wild-type (WT) p53 allele were significantly more radioresistant than cell lines which lacked a WT gene. We observed nine HNSCC cell lines with known intrinsic radiosensitivity and p53 allele status with time-lapse video microscopy after irradiation with 2 and 3 Gy. We studied the mitotic and apoptotic frequencies and scored the apoptoses as to whether they occurred morphologically in mitosis or in interphase. Irradiation with 2 or 3 Gy did not induce apoptosis in the cell lines studied. As expected the mitotic frequency was reduced by the irradiation. This was statistically significant in the cell lines which were radiosensitive when measured with a clonogenic assay. p53 allele status did not have an independent effect on the cell lines, except that the irradiation favoured the apoptosis in mitosis in the cell lines with WT p53 and the apoptosis in the interphase in the cell lines with a mutated or non-functional p53 gene. We conclude that although the apoptosis is not induced by irradiation with 2 Gy or 3 Gy, the p53 suppressor gene seems to influence the process of apoptosis after irradiation in the cell lines studied.

Apoptosis in situ, p53, bcl-2 and AgNOR counts as prognostic factors in laryngeal carcinoma.

Apoptotic cell death, in conjunction with mitosis, plays an important role in maintaining tissue homeostasis. Many cancer treatment modalities have been shown to induce apoptosis in sensitive cells. The purpose of this study was to determine the amount of apoptosis in-situ, the expression of apoptosis related genes p53 and bcl-2 and the proliferative marker nucleolar organiser region (AgNOR), in squamous cell carcinoma (SCC) of the larynx and correlate the results to clinical outcome. Paraffin-embedded samples of 66 patients with laryngeal SCC (34 glottic, 24 supraglottic and 8 transglottic) treated at Turku University Hospital were re-examined and divided into three histological grades of differentiation, four grades of keratinisation, and four grades of p53 and bcl-2 immunostaining. The apoptosis in-situ was assessed by TUNEL and was analysed as the number of apoptosis per volume corrected high power fields. The percentages of cells containing tumour nuclei with one to more than four AgNORs were counted. The patient median age was 65, the disease-free 5-year survival was 53% and the overall survival rate was 64%. In univariate analysis, nodal status, tumour size and general condition were associated with disease-free and overall survival. In a subgroup of patients with T1-2N0 glottic cancer receiving definitive irradiation therapy (n = 25), small tumour size and good histological differentiation were associated with good disease-free and overall survival. Apoptosis associated and proliferative markers did not seem to have more value to prediction of clinical outcome than the common clinical parameters.

Syndecan-1: a new prognostic marker in laryngeal cancer.

The cell adhesion molecule syndecan-1 expression is induced during keratinocyte differentiation and reduced during the formation of squamous cell carcinomas (SCCs). A significant correlation between decreased syndecan-1 expression in head and neck SCC measured from frozen sections with immuohistochemical methods and clinical outcome are reported. The clinical relevance of the cellular proliferation marker Ki-67 is controversial in SCC of the head and neck. The purpose of this study was to determine the expression of syndecan-1 and Ki-67 in SCC of the larynx and correlate the results with known prognostic factors and clinical outcome. Paraffin-embedded samples of 100 patients with laryngeal SCC (44 glottic, 36 supraglottic, 20 transglottic) treated at Turku University Central Hospital were re-examined and divided into four histological grades of differentiation, four grades of keratinisation, and four grades of 104-9 (syndecan-1) immunostaining. The mitotic index was analysed as the number of mitoses per volume corrected high power fields. The relative number of Ki-67 positive cells was evaluated. The patients mean age was 64 years and the 5-year survival was 69%. In univariate analysis, intermediate or strong staining for syndecan-1 was associated with higher overall survival than those tumours with no or little syndecan-1 expression (p = 0.048). Nodal status (p = 0.0001), tumour size (p = 0.0004) and localisation (p = 0.0008), general condition (p = 0.0001), histological grade (p = 0.02) and patient age (p = 0.03) correlated with overall survival whereas the Ki-67 index (p = 0.093), mitotic index (p = 0.23) and grade of keratinisation (p = 0.90) failed to do so. The results suggest that syndecan-1 could be a useful prognostic factor in SCC of the larynx.