Optical coherence tomography assessment of disease activity in cryopyrin-associated periodic syndrome.

BACKGROUND AND PURPOSE: Cryopyrin-associated periodic syndrome is a rare autoinflammatory disease caused by gain-of-function mutations or variants in the NLRP3 gene. Clinically, patients suffer from a broad spectrum of both systemic and neurological symptoms. The aim of this study was to determine whether systemic inflammation demonstrated by serum amyloid A (SAA) elevation is associated with neuroinflammation assessed by optical coherence tomography (OCT). METHODS: Thirty eyes of 15 patients with NLRP3 low penetrance mutations (PwNLRP3) and 20 eyes of 10 age- and sex-matched healthy controls were examined by spectral-domain OCT as part of routine clinical care. All retinal layers and clinical features were evaluated. RESULTS: At baseline no significant retinal neuroaxonal inflammation or degeneration was observed in all measured retinal layers amongst PwNLRP3 compared with healthy controls. In a pooled analysis of all individual OCT time points a significant difference regarding the macular retinal nerve fibre layer was detected. Increased levels of SAA showed a positive association with averaged combined outer plexiform layer and outer nuclear layer volumes (ρ < 0.0001, r2 = 0.35). CONCLUSION: In cryopyrin-associated periodic syndrome increased combined outer plexiform layer and outer nuclear layer volumes are mirrored by SAA increase, an acute phase reactant indicating systemic inflammation. Our findings identify OCT as a candidate biomarker to monitor subclinical neuroinflammation and to assess disease activity in PwNLRP3.

Influence of freeze-drying on the recovery of the tumour invasion markers uPA and PAI-1 from prostate cancer resections.

BACKGROUND: Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) have been shown to be of merit as biomarkers for a variety of cancers. Prostate tissue resections from patients with prostate cancer (PCa) and benign prostatic hyperplasia were analysed to determine the influence of freeze-drying on the recovery of uPA and PAI-1 and their predictive performance. METHODS: Prostate tissue was frozen in liquid nitrogen and homogenised into a fine powder in a precooled stainless steel punch homogeniser. One aliquot of the powder was extracted directly, and a second aliquot was freeze-dried overnight and then extracted. The extracts were analysed by FEMTELLE assay to determine the concentrations of uPA and PAI-1. uPA/PAI-1 ratios were calculated for each sample, and the mean ratios for the frozen and the lyophilised tissue were compared. RESULTS: The concentrations of uPA measured for the frozen and lyophilised samples are strongly correlated (R = 0.90 ± 0.05). The same applies to the PAI-1 measured (R = 0.89 ± 0.03). The uPA/PAI-1 ratios for the lyophilised and frozen samples were strongly correlated. The uPA/PAI-1 ratios for frozen and lyophilised samples were found to be essentially the same with values of 0.0344 ± 0.0066 and 0.0340 ± 0.0068, respectively (P = 0.9633). CONCLUSION: The recovery of uPA and PAI-1 from a deep frozen prostate tissue homogenate followed by freeze-drying proceeds with a loss of 10 and 11%, respectively, with no influence on the uPA/PAI-1 ratio. Lyophilisation is a safe procedure for the preservation of frozen prostate tissue samples as it permits recovery of the markers without compromising their use for diagnostic purposes.

Plasma sarcosine does not distinguish early and advanced stages of prostate cancer.

INTRODUCTION: Diagnosis of prostate cancer by prostate specific antigen (PSA) is error-prone and cannot distinguish benign prostatic hyperplasia (BPH) from malignant disease, nor identify aggressive and indolent types. METHODS: We determined serum sarcosine (N-methylglycine) in 328 cancer patients by gas chromatography (GC)/mass spectroscopy (MS) and searched for correlations with early (stage T1/T2) and advanced (stage T3/T4) disease. RESULTS: Serum sarcosine of male control patients ranged from 1.7 µmol/l to 4.8 µmol/l. In prostate cancer patients, sarcosine ranged from 2.8 µmol/l to 20.1 µmol/l. Expressed as the sarcosine/alanine ratio, serum control values were 9.4 ± 5.5 x 10(-3) (mean ± SD) compared with 21.6 ± 9.0; 28.5 ± 16.6; 22.7 ± 7.7 and 22.2 ± 11.0 for patients diagnosed with T1, T2, T3 and T4 prostate tumours, respectively. The small differences between T1, T2, T3 and T4 patients were not statistically significant (p=0.51). However, the conventional PSA marker significantly correlated with T stage in these patients (r=0.63; p<0.009). CONCLUSIONS: The median sarcosine/alanine ratios among patients with early and advanced prostatic cancer ranged from 21.6 ± 9.0 to 28.5 ± 16.6 and were fairly constant, showing no statistically significant differences between T-stages. The results are consistent with published data in urine and serum which find differences between controls and patients with metastatic prostate cancer to be small and sarcosine to be uninformative regarding prostate cancer progression. By multi-comparison of PSA with T-stages in the same group of patients, we found significant correlations confirming the well-known merits and limitations of this marker.

Influence of irinotecan and SN-38 on the irradiation response of WHO3 human oesophageal tumour cells under hypoxic conditions.

Irinotecan and its metabolite SN38 were evaluated for their cytotoxicity and influence on radiosensitivity in WHO3 human oesophageal cells under hypoxic conditions. The IC50's of Irinotecan and SN-38 were found to be 0.8 and 0.04 microM, repectively, with SN-38 emerging as the more potent drug. The toxicities were similar under anoxic conditions. Given in conjunction with irradiation under hypoxic conditions, the two drugs restored the radiosensitivity of WHO3 cells in a dose-dependent manner by factors of 1.5-2.1 as compared to a control oxygen enhancement ratio (OER) of 2.1 in this cell system. In the subtoxic concentration range of 10(-2) microM SN-38 still generated a marked sensitisation of hypoxic tumour cells by factors of 1.2-1.6. It is concluded that the topoisomerase inhibitor Irinotecan and in particular the metabolite SN-38 may be clinically useful for radiotherapy of notoriously hypoxic tumour pathologies.

Evaluation of cisplatin and a novel platinum polymer conjugate for drug toxicity and drug distribution in mice.

The toxicity and distribution of cisplatin and two novel platinum (Pt) polymer conjugates (Pt-6 and Pt-7) were determined in serum and tissue of BALB/c mice at specific time points after i.p. administration of a drug bolus containing identical Pt concentrations. Pt concentrations were determined in serum, liver and kidney at 5 and 15 min, respectively, after drug administration by inductively coupled plasma mass spectrometry. It was found that the Pt polymer Pt-7 gave rise to a considerably lower Pt concentration in serum and considerably higher concentration in liver and kidney than cisplatin. LD25 measurements indicated that the Pt-7 polymer is considerably less toxic than cisplatin. In vitro experiments and determination of IC50 values in a variety of human tumor cell lines, normal lymphocytes and fibroblasts confirmed that Pt-6 and Pt-7 polymers are 40-500 times more toxic for tumor cells than for normal cells, perhaps reflecting preferential uptake. The toxicity of cisplatin was found to be only 1.6-40 times more effective in tumor cells. These inter-relationships are supported by the observation that the tumor enrichment factor (TEF) for cisplatin is only in the region of 6, and much lower than for Pt-6 and Pt-7, where TEFs are in the region of 40 and 150, respectively. These results demonstrate that the Pt polymer conjugates exhibit greater tumor specificity than cisplatin, killing tumor cells more effectively while being considerably less toxic for normal cells. It is concluded that the Pt polymer conjugates may be superior for cancer therapy and warrant further testing to assess their full clinical potential.

Influence of DNA double-strand break rejoining on clonogenic survival and micronucleus yield in human cell lines.

PURPOSE: To examine the role of DNA double-strand break (DSB) rejoining in cell survival and micronucleus yield after 60Co gamma-irradiation. MATERIALS AND METHOD: Thirteen human cell lines (six glioblastoma, five prostate, one melanoma, one squamous cell carcinoma) were irradiated with 60Co gamma-rays to doses of 0-10Gy for cell survival and micronucleus measurements and 0-100Gy for DSB rejoining. Measurements were performed using standard clonogenic, micronucleus and constant-field gel electrophoresis assays. RESULTS: Radioresistance and micronucleus yield were positively correlated (r=0.74, p=0.004). A significant cell type-dependent correlation was demonstrated between total (0-20 h) DSB rejoining and cell survival (r=0.86, p=0.03 for glioblastomas; r=0.79, p=0.04 for other cell lines), with more resistant cell lines showing higher levels of DSB rejoining. No relationship was apparent between fast (0-2 h) or slow (2-20 h) DSB rejoining and clonogenic survival. While there was no relationship between total or slow DSB rejoining and micronucleus yield, a significant and cell type-specific correlation emerged between fast rejoining and micronucleus yield for the glioblastomas (r=0.89, p=0.04) and other cell lines (r=0.76, p=0.04). Cell lines with higher levels of DSB rejoining within 2 h of irradiation showed higher yields of micronuclei. CONCLUSION: Fast DSB rejoining, possibly through interaction with slow DSB rejoining, appears to play an important role in the formation of micronuclei. However, total DSB rejoining reflects intrinsic radiosensitivity. Consideration of differences in DSB rejoining kinetics might contribute to a better understanding of the significance of cell survival and micronucleus data in the clinical and radiation protection setting.

Flow cytometric evaluation of apoptosis and cell viability as a criterion of anti-tumour drug toxicity.

BACKGROUND: Determination of the drug concentration required to kill 50% of the tumour cells (EC50) does not take into account the propensity of cells to undergo apoptosis and necrosis. These 2 parameters and the viable cells are here assessed by a flow cytometric (FC) approach using propidium iodide (PI) and FITC-Annexin V staining. MATERIALS AND METHODS: A number of carcinoma cell lines of defined p53 status were exposed to cis-PtII for 24 hours, stained with PI and FITC-Annexin V and analyzed by FC. Unstained viable cells, early apoptotic cells and necrotic cells were scored separately in dual parameter plots of green fluorescence (FITC) against red fluorescence (PI) to generate dose response curves. RESULTS: EC50 values for cell viability were found to be 1-4 times higher than survival data from colony assays resembling data obtained by MTT or Crystal Violet vital dye staining. Percentage apoptosis measured by Annexin V binding was in agreement with microscopic scoring of apoptotic cells after Acridine Orange staining. CONCLUSION: The FC assay described gives a good estimate of cell viability resembling data from vital dye staining assays and provides additional information on apoptosis and necrosis. FC data from Annexin V binding and microscopic scoring after Acridine Orange staining were in excellent agreement.

Radiosensitization and DNA repair inhibition by pentoxifylline in NIH3T3 p53 transfectants.

PURPOSE: To examine the role of p53 mutations in the modulation of DNA repair and radiotoxicity by pentoxifylline. MATERIALS AND METHODS: NIH3T3 murine cells transfected with mutant p53 constructs were examined for the influence of pentoxifylline on radiotoxicity to Co(60) gamma-irradiation by colony assay. DNA repair (0-100 Gy) was measured by constant-field gel electrophoresis. Apoptosis was assessed by flow cytometry with the annexin-V-binding assay. RESULTS: In the two p53 hot-spot mutant cell lines p53-S269R and p53- + 15, the SF(10) radiotoxicity enhancement factors induced by the pentoxifylline were 8.0 and 9.7, respectively. In the p53 deletion mutant p53-DeltaA cell line, the radiotoxicity enhancement factor was 2.6. No radiosensitization was obtained in the untransfected p53 wild-type cell line U-Wt and in the transfected p53 double-wild-type p53-Wt cell line. When pentoxifylline was added after irradiation at the time of maximum G2 block expression, no radiosensitization was observed in any of the five cell lines. Constant-field gel electrophoresis analyses after 20 h of repair showed that pentoxifylline suppresses DNA double-strand break repair in all p53 mutant cell lines, as indicated by repair inhibition factors of 2.0-2.3. No repair suppression was found in the p53 wild-type cell lines. CONCLUSIONS: p53 mutations are a general requirement for radiosensitization by pentoxifylline and the level of radiosensitization depends upon the location of the p53 mutation.

The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation 1-10 Gy.

Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence. Cell survival curves of irradiated CHO-K1 cells showed that treatment with nocodazole increases radiosensitivity as indicated by a decrease of the mean inactivation dose (D) from 4.446 to 4.376. Nocodazole treatment increased the initial radiation-induced DNA damage detected by the FADU assay from 7% to 13%. In repair-defective xrs1 cells, the same conditions increased the radiosensitivity from 1.209 to 0.7836 and the initial DNA damage from 43% to 57%. Alterations to chromatin structure by hypertonic medium increased radiosensitivity in CHO-K1 cells from of 4.446 to 3.092 and the initial DNA damage from 7% to 15%. In xrs1 cells these conditions caused radiosensitivity to decrease from 1.209 to 1.609 and the initial DNA damage to decrease from 43% to 36%. Disruption of chromatin structure by hypertonic treatment was found to be time-dependent. A threefold increase of exposure time to hypertonic medium from 40 to 120 min increased the initial DNA damage in CHO-K1 cells from 7% to 18% but decreased initial DNA damage in xrs1 cells from 43% to 21%. Perturbation of chromatin structure with hypertonic treatment has been shown to increase the radiosensitivity and the initial DNA damage in repair-competent CHO-K1 cells and decrease the radiosensitivity and DNA damage in repair-defective xrs1 cells. Hypertonic treatment thus abolishes differences in chromatin structure between cell lines and differences in initial DNA damage. Radiosensitivity and initial DNA damage are correlated ( r(2)=0.92; p=0.0026) and this correlation also holds when chromatin compaction is altered. The experiments demonstrate that initial DNA damage and chromatin structure are major determinants of radiosensitivity.

The effect of radiation on the permeability of human saphenous vein to 17 beta-oestradiol.

Radiation therapy is an effective way of treating many forms of cancer, however, there are some indications that it may facilitate the development of metastasis. The question arises whether radiation therapy during cancer treatment might result in an alteration of the permeability of the tissues being treated. This alteration in the permeability might lead to metastatic cells escaping from the irradiated tissue, leading to the spread of cancer to other sites in the body. Because of the above implication, we determined the diffusion kinetics of a radioactive marker, 17 beta-oestradiol, through human saphenous vein before and after a single half hour exposure to 60 Gy of 60Co gamma-irradiation. Six clinically healthy saphenous vein specimens (mean patient age +/- standard deviation 57 +/- 13 years; age range 41-77 years) were obtained during cardiac surgery. In vitro flux rates of 17 beta-oestradiol were determined through use of a flow-through diffusion apparatus immediately after irradiation for a period of 24 hours. No statistically significant differences could be demonstrated for the flux rates of 17 beta-oestradiol through the non-irradiated and 60 Gy irradiated saphenous vein tissue. These findings strongly suggest that irradiation at 2 Gy/min and a total dose of 60 Gy would not alter the permeability of the venous wall. We have demonstrated that the in vitro flow-through diffusion method is capable of measuring permeability aspects of endothelial cell layers in saphenous vein biopsies under conditions resembling clinical reality.

Influence of irradiation and pentoxifylline on histone H3 phosphorylation in human tumour cell lines.

Phosphorylation of histone H3 at Ser-10 correlates with chromatin condensation and this amino terminal modification is now recognized as a specific marker of mitosis. We have monitored the appearance of cells showing histone H3 phosphorylation in four human tumour cell lines to identify cell cycle progression after irradiation. In the human melanoma cell lines Be11 and MeWo and in the squamous cell carcinoma lines 4197 and 4451 a dose of 7 Gy of Co-gamma irradiation increases the number of cells binding anti-histone H3-P antibody 1-8-fold in a p53-independent manner. In the p53 mutant cell lines MeWo and 4451 H3-P phosphorylated cells can be detected as early as 30 min and show a maximum 1 h post-irradiation. In the cell lines Be11, 4197 and 4451 the early wave of H3 phosphorylated cells is followed by a second wave, which reaches a maximum 4.5-7 h post-irradiation and then declines. These events are attributed to damage-induced cell cycle blocks in the G1 and G2 phase of the cell cycle. Addition of the dose modifying drug pentoxifylline before irradiation increases the appearance of cells showing early and the late H3 phosphorylation. When pentoxifylline is added 12-24 h post-irradiation when the cell cycle blocks have reached their maximum the appearance of cells with phosphorylated H3 increases 3-5-fold in the p53 mutant cell lines MeWo and 4451. These observations are consistent with the function of the drug as a G2 block abrogator. The large H3 phosphorylation signal in p53 mutant cells is consistent with early entry of a cohort of G2 cells into mitosis. The smaller H3-P signal in p53 wild type cells correlates with the lower proportion of stable G2 populations in G1 blocked cells. These results indicate that pentoxifylline influences the appearance of histone H3 phosphorylated cells in a manner strongly dependent on the number of cells in G2 phase. This suggests that addition of pentoxifylline indeed abrogates the G2 block and thereby facilitates early entry into mitosis.

A unifying model for reconstructing radiosensitivity from micronucleus formation, apoptosis and abnormal morphology.

At present micronucleus data cannot predict cellular radiosensitivity. The inclusion of data from apoptosis and abnormal morphology has not entirely resolved this problem. Here, we assess the probability of cell death arising from events other than micronucleation, apoptosis and abnormal morphology (i.e. lesions not detected by these damage assays) P(oe), for its ability to reflect intrinsic cellular radiosensitivity. Analysis of data from 17 cell lines used in two separate studies, spanning a wide range of radiosensitivity (0.09

Proton RBE for early intestinal tolerance in mice after fractionated irradiation.

BACKGROUND AND PURPOSE: To determine the influence of the number of fractions (or the dose per fraction) on the proton relative biological effectiveness (RBE). MATERIALS AND METHODS: Intestinal crypt regeneration in mice was used as the biological endpoint. RBE was determined relative to cobalt-60 gamma rays for irradiations in one, three and ten fractions separated by a time interval of 3.5h. Proton irradiations were performed at the middle of a 7-cm Spread Out Bragg Peak (SOBP). RESULTS: Proton RBEs (and corresponding gamma dose per fraction) at the level of 20 regenerated crypts per circumference were found equal to 1.15+/-0.04 (10.0 Gy), 1.15+/-0.05 (4.8 Gy) and 1.14+/-0.07 (1.7 Gy) for irradiations in one, three and ten fractions, respectively. Alpha/beta ratios as derived from direct analysis of the 'quantal radiation response data' were found to be 7.6 Gy for gamma rays and 8.2 Gy for protons. Additional proton irradiations in ten fractions at the end of the SOBP were found to be more effective than at the middle of the SOBP by a factor of 1.14 (1.05-1.23). CONCLUSION: Proton RBE for crypt regeneration was found to be independent of fractionation up to ten fractions. One can expect that it remains unchanged for higher number of fractions as the lethalities for doses smaller than 3 Gy are exclusively due to direct lethal events. As a tendency for increased effectiveness at the end of the SOBP is reported in the majority of the studies, for clinical applications it would be advisable to allow for by arranging a sloping depth dose curve in the deeper part of the target volume. Finally, it must be noticed that most of in vitro and in vivo RBE values for protons are larger than the current clinical RBE (RBE=1.10).

Chemosensitivity of prostatic tumour cell lines under conditions of G2 block abrogation.

Conventional chemotherapy has had very limited success in the control of hormone-refractory prostate cancer. Methylxanthine derivatives, such as pentoxifylline (PTX), are known to abrogate the G2 block and enhance the toxicity of ionising irradiation and chemotherapeutic agents. It is now also established that late addition of the cytotoxic drug after irradiation under conditions of G2 block abrogation sensitises human tumour cells for cytotoxins. Here we assess whether the chemosensitivity of prostate tumour cell lines can be enhanced by the application of a low dose of drug in conjunction with a G2 block abrogator. Prostate cell lines DU145, BM1604 and LNCaP were irradiated with 7 Gy 60Co gamma-irradiation. A sub-toxic (2 mM) dose of pentoxifylline and a cytotoxic drug were added at maximum expression of the G2 cell cycle block and cell survival was determined by colony assay. Cisplatin, etoposide and vinblastine were tested at a toxic dose of 10% (TD10). In the TP53 mutant cell lines, DU145 and BM1604, dose enhancement factors (EFs) were found to be in the region of 4.20 for cisplatin, 3.70 for vinblastine, and 3.20 for etoposide. In the TP53 wild-type cell line, LNCaP, the enhancement factors were low and in the region of 1.20 for cisplatin, vinblastine and etoposide. It is clear, therefore, that toxicity enhancement factors (EFs) are greater in the TP53 mutant cell lines, DU145 and BM1604, than in the TP53 wild-type cell line, LNCaP. The results indicate that a significant enhancement of drug toxicity can be obtained if the cytotoxic drug is given under conditions of G2 block abrogation. The sensitisation of prostate cancer cells to cytotoxic drugs is particularly high in radiation-resistant TP53 mutant tumour cells. Drugs which abrogate G2 block have the potential to enhance the therapeutic index and therefore reduce the toxicity of chemotherapy drugs.

Comparison of cell inactivation by Auger electrons using the two reagents 4-[123I]iodoantipyrine and [123I]NaI.

The Auger electron emitter 123I was examined in the form of 4-[123I]iodoantipyrine and as [123I]NaI for its effectiveness in killing cells of different sensitivity to photon irradiation. Micronucleus assays showed that 4-[123I]iodoantipyrine is 2-3 times more effective in cell inactivation than [123I]NaI. This can be attributed to the fact that antipyrine, for reason of its lipid solubility, can enter cells and can reach the nucleus, whereas [123I]NaI is excluded from the cytoplasm. In the nucleus Auger decay is conceivably located on the DNA where it may invoke high-LET irradiation damage. Irradiation damage by [123I]NaI is by long range Auger and internal conversion electrons and hence less densely ionising. Results of the present study demonstrate, however, that the enhancement of micronuclei frequency (MNF) seen with 4-[123I]iodoantipyrine as compared to [123I]NaI is similar for all cell lines and that the ratio of 4-[123I]iodoantipyrine/[123I]NaI MN response remains the same. Experiments with the free radical scavenger DMSO, indicated nearly identical dose reduction factors for both 123I carriers. These two observations strongly suggest that the cell inactivation by 4-[123I]iodoantipyrine is not by direct high-LET ionisation of DNA, but is due to an indirect effect. The indirect radiation effect of Auger decay in the nucleus could arise because 4-[123I]iodoantipyrine is not incorporated into the DNA, but is only associated with chromatin where the DNA is shielded by histones.

Cytotoxicity of azadirachtin A in human glioblastoma cell lines.

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.

Micronuclei and apoptosis in glioma and neuroblastoma cell lines and role of other lesions in the reconstruction of cellular radiosensitivity.

It is now well established that micronuclei frequency does not always rank cell lines according to radiosensitivity. There is, however, a growing interest in reconstructing cellular radiosensitivity (measured by colony assay) from concurrent micronucleus and apoptosis data. Using a variety of radiosensitive and radioresistant cell lines, we have derived a missing parameter--Poe, the probability of cell death by other events such as small deletions, chromosome aberrations, late apoptosis and necrosis which are undetectable by micronucleus and apoptosis assays performed at a single time point. In the radioresistant glioma cell lines G120, G60, G28, G44 and G62 (SF2 > or =0.59), a characteristic threshold dose exists above which cell loss due to undetectable lesions occurs. In the radiosensitive SK-N-SH and KELLY cell lines (SF2 < or =0.43), the Poe parameter is positive at very low doses, reaches a maximum and declines at higher doses. In the radiation resistant G28 cells, Poe was found to be below zero for doses up to 6 Gy. In the G62, G44 and G120 cell lines, the threshold doses to induce Poe events were 0.87, 3.04 and 3.85 Gy, respectively. Cell death by undetectable lesions is a cell-specific and time-dependent variable. Micronucleus and apoptosis assays performed concurrently and at a specific time point miss cell death due to other events and this may be the reason why reconstruction of cellular radiosensitivity from micronucleus and apoptosis data fails.

Influence of pentoxifylline, A-802710, propentofylline and A-802715 (Hoechst) on the expression of cell cycle blocks and S-phase content after irradiation damage.

The toxicity of the five methylxanthine derivatives, caffeine, pentoxifylline, A802710, propentofylline and A802715, was determined against the two human melanoma lines, Be11 and MeWo, and against the two human squamous cell carcinoma lines, 4197 and 4451, by vital dye staining assay. Pentoxifylline and A802710 emerge as the least toxic showing TD(50) (toxic dose of 50%) levels of 3.0-4.0 mM. Propentofylline and caffeine take an intermediate position. A802715 has a TD(50) of 0.9-1.1 mM and is the most toxic. Subtoxic concentrations (

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation.

PURPOSE: To determine the relationship between DNA damage and radiosensitivity at low doses (1-10 Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. MATERIALS AND METHODS: Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60Co gamma-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). RESULTS: The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2 = 0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. CONCLUSION: It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.