Kirsi Pekkola-Heino

In Vitro Radiation Resistance Among Cell Lines Established From Patients With Squamous Cell Carcinoma of the Head and Neck

Twenty‐five squamous cell carcinoma (SCC) cell lines from 20 patients with head and neck cancer were assessed for radiosensitivity in vitro using a 96‐well plate assay. Four non‐SCC lines were also tested. Radiation sensitivity of individual cell lines was compared using the area under the survival curve (AUC) as a measure of the mean inactivation dose. Tumor lines were tested with either a cobalt‐60 (60Co) γ‐irradiator having a dose rate of 100 cGy/minute or with a 4‐meV photon beam having a dose rate of 200 cGy/minute. The mean AUC of the 25 SCC cell lines was 188 ± 7 (SEM) cGy (range, 100 to 250 cGy) whereas the four non‐SCC lines had a mean AUC of 225 ± 9 cGy. The SCC cell lines with mean inactivation dose values greater than 188 cGy were classified as relatively radioresistant whereas those with values less than 188 cGy were considered relatively radiosensitive. In seven cases SCC cell lines were derived from patients who had already received radiation therapy. In four of these cases the tumor cell lines were radioresistant (AUC, 210 to 250) but in the other three cases the tumor lines were radiosensitive (AUC, 160 to 180). Thus, failure of a tumor to respond to radiation did not always select for radioresistant cells. The mean of the AUC for cell lines from previously irradiated patients (197 ± 11 cGy) did not differ significantly from that of the cell lines from patients who received no prior radiation therapy (182 ± 9 cGy). However, among radiation‐resistant lines those from the four previously irradiated patients were significantly more resistant (mean AUC = 235 ± 9) than seven other radioresistant lines from nonirradiated patients (mean AUC, 208 ± 4) (P = 0.0194). In four cases more than one cell line was derived from different tumor specimens in the same patient. In each of these cases the lines from the same patients were similar to one another in their degree of radioresistance. Based on these observations the authors conclude that the degree of in vitro radiation resistance is an inherent property of some squamous cell tumors.

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.

Effects of radiation fractionation on four squamous cell carcinoma lines with dissimilar inherent radiation sensitivity.

SummaryThe effect of radiation fractionation was investigated using a new 96-well-plate clonogenic assay in four squamous cell carcinoma lines. Earlier experiments had shown that two of the cell lines (UT-SCC-1A and UM-SCC-14A) were inherently relatively sensitive to irradiation, and two (UM-SCC-1 and UM-SCV-1A) relatively resistant. All of the four carcinomas from which the cell lines were established had poor clinical outcome. The radiation doses were given as a single exposure, or split into two or three equal fractions with a 24-h interval. The two inherently sensitive cell lines showed enhanced survival after radiation fractionation as compared with a single dose, whereas the resistant cell lines did not. The result suggests that both the inherent resistance of cancer cells to irradiation and the repair of sublethal radiation damage may lead to treatment failure, and that shortening of the total irradiation time may overcome cancer cell recovery between fractions in some, but not in all carcinomas.

Increased radiosensitivity is associated with p53 mutations in cell lines derived from oral cavity carcinoma

The curability of oral cavity carcinomas, as well as of other head and neck cancers, varies remarkably especially in more advanced disease. Radiotherapy and surgery, including large operations, are currently combined, but as new radiotherapy regimens are being introduced, the need for predictive assays has increased in order to plan a suitable individual treatment for the patient. Variations in intrinsic radiation sensitivity of cancer cells cannot alone explain differences in therapy outcome, and thus additional predictive variables have to be searched. Mutations in the p53 tumor suppressor gene are found in most head and neck tumors, which has led us to study the possible association between these mutations and radiation sensitivity. We analyzed 16 cell lines from oral cavity carcinomas and found a remarkable variation in radiosensitivity (AUC 1.7-2.3 Gy and SF2 0.31-0.51). The p53 gene was mutated in 11/16 cell lines, and these cells were also significantly more sensitive than those with wildtype p53 (AUC 1.9 +/- 0.2 Gy and 2.3 +/- 0.2 Gy, respectively, p = 0.023). Structural alterations in the p53 gene were also observed in three of the relatively resistant cell lines, which indicates that not all mutations are critical in this respect.

Comparison of cellular radiosensitivity between different localizations of head and neck squamous-cell carcinoma

The prognosis of carcinomas arising from various sites in the head and neck varies even when the stage of the disease is taken into consideration, e.g. laryngeal carcinoma has a more favourable prognosis compared to oral-cavity malignancies. The purpose of this study was to evaluate intrinsic cellular radiosensitivity as one possible explanation for the observed differences in the survival rates of different anatomical groups. The radiation survival curves were determined for well characterized cell lines derived from laryngeal carcinoma (n=14), pharyngeal carcinoma (n=6), carcinoma of the oral cavity (n=14) and the skin of the face (n=3). The intrinsic radiosensitivity was expressed as area under the survival curve (AUC) values, and this cellular parameter was compared with clinical data and survival of the patients. The intrinsic radiosensitivity in the whole group varied between 1.0 Gy and 2.8 Gy with an average of 1.9 Gy. The mean AUC values for the laryngeal cell lines were 2.0 Gy±0.2, for the oral cavity 1.8±0.3 Gy, for the pharynx 1.8±0.2 Gy and for cutaneous carcinoma 2.1±0.1 Gy. There was a slight difference between the groups of glottic and supraglottic cell lines (mean 1.8±0.2 Gy and 2.1±0.3 Gy, respectively), which is consistent with the differences in clinical curability of these cancers. Otherwise, the differences in cellular radiosensitivity of the carcinoma groups studied did not reach statistical significance. These results indicate that the intrinsic radiosensitivity of squamous-cell carcinoma (SCC) of the larynx does not significantly differ from that of SCC of other sites of the head and neck. Variations in the intrinsic radiosensitivity do not as such seem to explain the observed differences in radiocurability of SCC variously localized in the head and neck.

Sublethal damage repair capacity in carcinoma cell lines with p53 mutations.

The previous findings that sublethal damage repair (SLDR) capacity varies between carcinoma cell lines and that the inherent radiosensitivity of these lines tends to be higher in connection with p53 mutations lead us to study the possible role of p53 gene in the regulation of SLDR. The activation of p53 gene by irradiation is known to cause changes in cell cycle progression. Thus, p53 status probably has effects on cellular radiosensitivity, theoretically through modulating repair processes.

The bcl-2 Gene Status of Human Head and Neck Cancer Cell Lines

The bcl-2 oncogene was originally found in the translocation in a pre-B cell acute lymphocytic leukemia cell line. Since then a high expression of Bcl-2 has been found in many types of cancer. The bcl-2 gene encodes an intracellular membrane-associated protein. Overexpression of bcl-2 inhibits apoptosis induced by many drugs and radiation. In this study the bcl-2 gene status of 9 human head and neck squamous cell carcinoma cell lines was studied. Mutations of the bcl-2 gene were studied at mRNA and DNA levels. The presence and abundance of the Bcl-2 protein in cells were also investigated. In earlier studies the p53 tumour suppressor gene was screened for point mutations, and the radiosensitivity of these cell lines was measured. We were able to amplify bcl-2 cDNA from 5 of the 9 cell lines, which shows that bcl-2 was expressed in these cells. No point mutations were found in the bcl-2 gene in any of these cell lines. Loss of heterozygosity was observed in 2 cell lines at the bcl-2 locus, and these cell lines had no detectable levels of bcl-2 mRNA or Bcl-2 protein. The Bcl-2 protein was abundant in the cell lines with the wild-type p53 gene, and these cell lines were radioresistant. The Bcl-2 protein was also found in many other cell lines in mitotic cells. It seems that cells expressing bcl-2 are radioresistant, and even functional p53 cannot induce apoptosis in these cells.

DOSIMETRY OF IRRADIATION MODELS The 96-well clonogenic assay for testing radiosensitivity of cell lines

Radiation experiments with cells in single cell suspension in test tubes and on 96-well plates were carried out and compared. The cells originated from cell lines established from carcinomas of the floor of the mouth and from endometrial carcinoma. Two irradiation models were constructed. Both models allowed the absorbed doses to the cells to be administered with a high accuracy in both experimental settings (better than 5.0%). These irradiation models were compared on cancer cell lines with dissimilar inherent radiation sensitivity and histologic type (UM-SCC-1 resistant, UM-SCC-14A sensitive, and UT-EC-2B highly sensitive); various radiation doses were used. The fractions of surviving cells as a function of radiation dose were compared: there was no significant difference between cells irradiated in test tubes and cells irradiated in 96-well plates. Thus, if the absorbed doses in cells suspended in a tube and in a plate were the same, the survival was similar regardless of the type of irradiation model.