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

BACKGROUND: 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. METHODS: The SDLR capacity of 17 head and neck carcinoma cell lines was determined in split-dose experiments using a 96-well plate clonogenic assay. The SLDR capacity as well as the inherent radiosensitivity were compared with the p53 status of the cells. RESULTS: The SLDR capacity varied markedly also between cell lines of similar radiosensitivity, but there was a tendency of the more sensitive cells to be more SLDR proficient .(r = -.69; p = .0016). The (beta-values obtained from linear quadratic equation correlated well with the observed amount of SLDR (r = .73; p = .0006). With one exception, those cell lines having p53 mutations showed higher SLDR than those with no mutations (p = .0017). In many of these cell lines, the mutations caused either total loss of the p53 protein or major, probably functional changes in it. The cell line UT-SCC-16A, showing no SLDR in the experiments, had two mutation points in different alleles, perhaps having less effect on the protein function. CONCLUSION: This extended material confirmed the previous result that the SLDR capacity tends to increase with increasing radiosensitivity in carcinoma cell lines. A clear correlation between p53 mutations and SLDR capacity was found. The SLDR depended, however, on loss of normal p53 function, which implies that the p53-mediated G1 arrest is not as important in this repair process, as would have been expected.

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.

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.

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.

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.

Cytotoxicity of bleomycin and external radiation in squamous cell cancer cell lines of head and neck.

Bleomycin, a natural antibiotic toxic to dividing cells, has been successfully used in combinations for treatment of recurrent head and neck cancer. In this study, we investigated five recently established head and neck squamous cell cancer lines (UT-SCC-2, UT-SCC-8, UT-SCC-9, UT-SCC-12A, UT-SCC-19A) in terms of response to bleomycin and radiation treatment. Experiments were carried out using the 96-well plate clonogenic assay for concentration determinations causing 20% (IC20), 50% (IC50), and 90% (IC90) inhibition of clonogenic survival and dose-response calculations for bleomycin. Survival fraction after a radiation dose of 2 Gy (SF2) was used as a measure for radiation sensitivity. The sensitivity for bleomycin was in good accordance with radiation sensitivity except for cell line UT-SCC-9. This was the cell line most sensitive to radiation (SF2 = 0.25 +/- 0.03) but was fairly resistant to chemotherapy (IC50 = 11.5M). Sensitivity patterns may suggest partly different mechanisms of action.

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.

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.

Relation of DNA ploidy and proliferation rate to radiation sensitivity in squamous carcinoma cell lines.

OBJECTIVE: According to some clinical reports, DNA aneuploidy of squamous cell carcinomas is related to a more favorable response to radiotherapy than DNA diploidy, but it is not known whether this depends on differences in intrinsic cellular radiation sensitivity or on other factors involved during the course of radiotherapy. DESIGN: Using flow cytometry, we analyzed 30 newly established squamous carcinoma cell lines derived from head and neck carcinoma, and compared the nuclear DNA content with the inherent radiosensitivity and with the average doubling time determined from the in vitro growth curves. RESULTS: The nuclear DNA content expressed as the ratio between the Go/G1 peak of the cell line and the peak formed by chicken red blood cells varied from 3.13 to 6.41. The DNA content of cells obtained from nonmalignant lesions by fine-needle aspiration biopsy was from 2.66 to 2.80. No correlation was found between the nuclear DNA content and the inherent radiosensitivity of the carcinoma cells. CONCLUSIONS: The result suggests that the intrinsic cellular radiosensitivity of carcinomas does not increase in parallel with an increasing nuclear DNA content. Nor does the in vitro proliferation rate associate with either the nuclear DNA content or inherent radiosensitivity.

Carboplatin-radiation interaction in squamous cell carcinoma cell lines.

Chemoradiotherapy has been considered one of the most promising improvements in the treatment of advanced head and neck cancer. This article describes in vitro chemosensitivity to carboplatin in five squamous cell carcinoma cell lines established from head and neck cancers and in one vulvar squamous cell carcinoma cell line. Sensitivity to carboplatin was found to vary markedly when using the 96-well plate clonogenic assay and continuous drug exposure. The difference in carboplatin response between the most sensitive and the most resistant cell lines was fourfold. No cross-resistance was observed between inherent radiosensitivity and chemosensitivity. Effects of concomitant use of carboplatin and radiation were further investigated in the two cell lines that were found to be most sensitive to carboplatin. The drug was administered 1 hour before acute radiation doses, and an additive effect was observed in both cell lines.

UT-MUC-1, a new mucoepidermoid carcinoma cell line, and its radiosensitivity.

Mucoepidermoid carcinoma is a fairly common malignant disease of the salivary glands. It is usually composed of two cell types, mucus-producing mucous cells and cells that have squamous differentiation with or without keratinization. The treatment of choice is considered to be radical surgical removal of the tumor. Radiotherapy has been recommended for high-grade or advanced tumors. Information on the radiosensitivity of mucoepidermoid cancer is scant, but based on clinical experience, the cancer appears to be only moderately radiosensitive. UT-MUC-1 is a newly established cell line from a poorly differentiated mucoepidermoid carcinoma. The cell line is diploid, but with complex karyotype abnormalities, giving a similar DNA index when measured with flow cytometry as that from the original tumor, and produces a tumor with characteristics typical for mucoepidermoid cancer when heterotransplanted to nude mice. The cell line is radiation resistant, with an area under the curve of 2.30 + 0.21 Gy when measured with a 96-well plate clonogenic assay. This correlates with the clinical outcome of the patient and the clinical experience of the radiosensitivity of this tumor type.

Sublethal damage repair in squamous cell carcinoma cell lines.

Squamous cell carcinoma (SCC) cell lines recently established from head and neck tumors were studied for their capability of repairing sublethal radiation induced damage (SLDR). Total doses of 1.25, 2.50, 5.00, and 7.50 Gy were used either as a single dose or split in two equal fractions with a 24 h interval. Cell survival was determined using a 96-well plate clonogenic assay based on limiting dilution. Survival data was fitted by linear quadratic model, and the area under the survival curve (AUC) was obtained with numerical integration. The amount of SLDR was expressed as AUC-ratio comparing survivals from split dose vs single dose experiments. SLDR capacity was observed to vary markedly between individual cell lines (AUC-ratios 1.0-1.5). The relatively radiation-sensitive cell lines had a tendency toward higher SLDR proficiency (correlation coefficient 0.85). The differences in repair capacity could not be explained by the differences in doubling times of the cell lines. The inverse relationship between SLDR capacity and inherent radiosensitivity could explain the poor radiocurability of the sensitive donor tumors. Two of the most resistant cell lines were found SLDR deficient. This is a novel finding, since SLDR has been previously reported in all studied cells.

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) gamma-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.

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

The 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.

Radiation response of vulvar squamous cell carcinoma (UM-SCV-1A, UM-SCV-1B, UM-SCV-2, and A-431) cells in vitro.

Standard therapy for squamous cell carcinoma (SCC) of the vulva consists of radical surgery and inguinal node dissection. Radiation therapy has been used for preoperative treatment in advanced cases to reduce the size of the tumor, and also as the only treatment in inoperable or recurrent disease. To study the inherent radiation sensitivity of vulvar carcinoma, we tested three new vulvar carcinoma cell lines and the long-established cell line A-431 by using a 96-well plate clonogenic assay, earlier shown by us to be suitable for survival studies of SCC. SCC and adenocarcinoma cell lines derived from other sites were used as a reference. Cells were irradiated with a 4-MeV linear accelerator at a dose rate of 2.0 Gy/min. The vulvar cell lines were found to be highly resistant to radiation with the average mean inactivation dose of 3.44 +/- 0.34 Gy as calculated from the area under the curve. The results were consistent in repeated experiments and for all cell lines. The average value for area under the curve was 1.79 +/- 0.30 for the other SCC lines tested. The values for area under the curve differed significantly (P less than 0.0001) between the vulvar lines and reference SCC lines. These results indicate that vulvar SCC cells in vitro express exceptional inherent radioresistance, and thus development of other forms of additional treatment would be more advantageous in advanced cases.

Circulating immune complexes during immunotherapy in allergy to dog.

Circulating immune complexes (CIC) were determined from dog-allergic asthmatic children (n = 35) receiving immunotherapy with dog dander and hair extract. The results from CIC are expressed in SDU (standard deviation units) and presented as follows: pretreatment results (n = 20), rush results (n = 11), mid-schedule results (n = 20), maintenance results (n = 15) and the results of the placebo-treated group (n = 12). The results of the placebo-treated group (n = 12) and those of the untreated atopic (n = 12) and non-atopic (n = 14) were controls. CIC levels were analysed by means of KgB-ELISA (conglutinin binding enzyme linked immunosorbent assay), C1qB-ELISA (C1q-binding enzyme linked immunososrbent assay), RFb-ELISA (rheumatoid factor binding enzyme linked immunosorbent assay) and by PIPA (platelet 125J-labelled staphylococcal protein-A test). The CIC level determined by KgB-ELISA in dog-allergic asthmatic children was higher than that of the atopic controls (P less than 0.05) already before the onset of the hyposensitization. During conventional hyposensitization with dog dander and hair the CIC level remained the same as before treatment. On day 5 of rush hyposensitization the mean level of CIC showed no increase when compared with the pretreatment values. A statistically significant correlation (P less than 0.01) was observed between the dog dander and hair-specific IgG antibodies and the CIC level measured by KgB-ELISA during the maintenance period of conventional immunotherapy. The samples of sera to measure this correlation were collected before the injection of allergen and after 2 weeks of injection during maintenance treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Yersinia antigens in synovial-fluid cells from patients with reactive arthritis.

We examined synovial-fluid cells from 15 patients with reactive arthritis after yersinia infection for the presence of yersinia antigens. Extensive bacterial cultures of the synovial fluid were negative. All the samples were studied by immunofluorescence with use of a rabbit antiserum to Yersinia enterocolitica O:3 and a monoclonal antibody to Y. enterocolitica O:3 lipopolysaccharide. Synovial-fluid cells from 41 patients with other rheumatic diseases served as controls. Synovial-fluid cells from 10 patients with reactive arthritis after yersinia infection stained positively on immunofluorescence; rabbit antiserum and the monoclonal antibody yielded similar results. In most patients the percentage of positive cells ranged from 1 to 10 percent, but in one patient nearly all the cells in the sample stained strongly. Most of the positively stained cells were polymorphonuclear leukocytes, but yersinia antigens were also found in mononuclear phagocytes. All the control samples were negative. Synovial-fluid cell deposits from nine patients were also studied by Western blotting with use of the same antibodies. The results were positive in six of the nine cell deposits from patients with reactive arthritis and in none of the 10 cell deposits from control patients with rheumatoid arthritis. We conclude that in patients with reactive arthritis after yersinia infection, microbial antigens can be found in synovial-fluid cells from the affected joints.

Role of antibodies in the opsonization of Yersinia spp.

We have determined the opsonic capacity of specific antibodies in patient sera obtained after Yersinia infection. The results indicate that Yersinia antibodies lead to complement activation through the classical pathway, thus overcoming the inhibition of complement-mediated opsonization in the absence of specific antibodies provided by the virulence plasmid in Yersinia enterocolitica and Yersinia pseudotuberculosis. Further, antibodies against plasmid-encoded structures, the Yersinia outer membrane proteins (YOPs), are not necessary in this effect. This is indicated by two facts. (i) Monoclonal antibodies directed against the O polysaccharide of Y. enterocolitica O:3 are capable of opsonizing the plasmid-containing bacteria through C1q binding. (ii) Rabbit antisera show opsonic activity when obtained by immunization both with plasmid-containing Y. enterocolitica expressing the YOPs and a plasmid-cured variant not expressing these proteins.

Immunoblotting analysis of human IgM, IgG and IgA response to chromosomally coded antigens of Yersinia enterocolitica 0:3.

Human antibody response after Yersinia enterocolitica infection was studied by immunoblotting sequentially collected sera against a whole-cell homogenate of Y. enterocolitica serotype 0:3, grown under conditions restrictive for the plasmid. The antibodies observed were directed against a multitude of chromosomally coded antigens, and a considerable individual heterogeneity was found in the reactions of individual sera. The early (0-2 months) and late (greater than or equal to 11 months) responses were directed against the same antigenic determinants. Antibodies against different bacterial epitopes decreased evenly with time, indicating that several, if not all, antigenic epitopes of the bacteria are responsible for the prolonged antibody production. IgM responses by most patients declined within a few months but were surprisingly strong in some even one year after onset of the infection. IgG antibodies showed a strong reaction against a region corresponding to lipid A and core of the bacterial LPS, whereas IgM and IgA recognized this region less often. No other significant differences between IgM, IgG and IgA responses were observed. Immunoblotting of sera from patients with post-infection complications (arthritis, iritis, erythema nodosum) did not reveal any additional or specifically involved antigens. Altogether, these findings suggest that Yersiniae causing the original infection may hide in some of the patients for prolonged periods.