Enhanced Radiation Sensitivity Research Articles

Nucleotide sequence analysis of c‐raf‐1 cDNA and promoter from a radiation‐resistant human squamous carcinoma cell line: Deletion within exon 17

The c-raf-1 proto-oncogene is the cellular homologue of v-raf, the oncogene of the acutely transforming retrovirus 3611-MSV. The product of c-raf-1 (raf-1) is a 74-kDa cytoplasmic serine/threonine protein kinase. We previously reported that antisense human c-raf-1 cDNA transfection results in reduction of the endogenous c-raf-1 transcript, decreased tumor growth rate, and enhanced radiation sensitivity of SQ-20B tumor cells established from a radiation-resistant laryngeal squamous cell carcinoma. In the study reported here, we used cDNA-linked polymerase chain reaction amplification and nucleotide sequencing to examine the structure of the 3233-bp SQ-20B c-raf-1 cDNA. The 812-bp c-raf-1 promoter region was analyzed by genomic DNA amplification followed by cloning and sequencing. Sequence comparison with a previously published c-raf-1 sequence indicated no structural changes within the coding region of SQ-20B c-raf-1. However, a 4-bp deletion was observed in the 3' untranslated region within exon 17. This deletion was also present in a c-raf-1 cDNA clone isolated from a SQ-20B cDNA library. While the possibility of a 3' transcriptional control mechanism cannot be ruled out, it appears that the raf-1 protein kinase may regulate the development of radioresistant malignancies via interaction with other molecules in the damage and repair-related signal transduction pathways.

Negative Supercoiling Increases the Sensitivity of Plasmid DNA to Single-strand Break Induction by X-rays

Negatively supercoiled topoisomers of the plasmid pIBI 30 were irradiated with 250 kV X-rays and assayed for strand scission by agarose gel electrophoresis. The survival of supercoiled molecules (Form I) decreased exponentially with increasing X-ray exposure and the dose required to reduce the fraction of DNA in Form I to 37% of its value in unirradiated controls (D37) decreased with increasing negative superhelicity. This enhanced radiation sensitivity of underwound DNA is tentatively attributed to the transient denaturation of the double helix that increases the susceptibility of individual strands to free radical attack.

Electrical and spin resonance characteristics of low-temperature plasma-enhanced chemical-vapor-deposited SiO2

Electrical and electron-spin-resonance measurements have been carried out on plasma-enhanced chemical-vapor-deposited SiO2 obtained using an N2O:SiH4 flow-rate ratio of 40 and a temperature of 300 °C. As-deposited films contain 2×1016 oxygen vacancy centers per cm3 while none were detectable in films annealed at 950 °C in Ar. 60Co γ radiation-induced defect creation is enhanced (>80 times for a dose of 20 Mrad) in as-deposited films with respect to thermal SiO2 consistent with the deposited film being in a state of densification (∼10%) with respect to undensified, fused silica. This result is in agreement with the results of density, refractive index, and infrared spectroscopy measurement. Annealed films, having a density close to thermal SiO2, are found to be very radiation sensitive in terms of positive fixed oxide charge creation but the relevant defects are not associated with paramagnetic centers. The origin of the enhanced radiation sensitivity in annealed samples may be related to the presence of incorporated nitrogen or excess silicon.

The relationship between radiation response of human squamous carcinoma cells and specific metabolic changes induced by chronic hypoxia

Chronic hypoxia can cause a range of metabolic changes within cells, such as increasing the synthesis rate of a group of proteins, oxygen regulated proteins (ORPs) and reducing glutathione (GSH) content. We are currently interested in the radiation responses of cells during recovery after chronic hypoxia in relation to these metabolic changes. Experiments have therefore been carried out on the influence of hypoxic pretreatment to y rays of two human squamous carcinoma cell lines, A431 (vulva) and CaSki (cervix), in exponential and plateau growth phases. In both cell lines, the synthesis rate of ORPs reached to a maximum level after 12 hr of hypoxia and during the same time, the cellular GSH content reduced by about 50%, but both returned to the control levels by 12 hr of reoxygenation. After 12 hr of hypoxic incubation at 37°C, cells were allowed to reoxygenate in air for 10 min (on ice) or 12 hr at 37°C before irradiation. Clonogenic assays were performed immediately after irradiation. Compared to the aerobic control, the radiosensitivity of both cell lines reoxygenated for 10 min after hypoxia increased significantly and later returned to the aerobic control level by 12 hr of reoxygenation. Since aerobic A431 cells treated with 10 mM buthionine sulfoximine for 12 hr did not increase the radiosensitivity, the enhanced aerobic radiosensitivity observed after chronic hypoxia was unlikely to be directly related to decreased GSH content. Further investigations of ORPs and other associated metabolic changes caused by chronic hypoxia have been in progress to determine their possible role in this enhanced radiation sensitivity.

Effect of Antisense c- raf -1 on Tumorigenicity and Radiation Sensitivity of a Human Squamous Carcinoma

Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer.

Enhancement of Radiation Sensitivity by Postradiation Hypoxia

We demonstrate that postradiation hypoxia during colony formation in vitro enhances radiation sensitivity when cells are irradiated in severely hypoxic states. The presence and magnitude of this phenomenon, if it occurs in vivo, raise questions about the importance of the oxygen effect in the radiation response of tumor cells in vivo and suggest that hypoxia may not be an important factor in the probability of tumor control following radiation therapy.

Iodized Cancer Therapy

Iodized Cancer Therapy

Enhanced Radiation Effects on Submicron Narrow-Channel NMOS

We have observed an enhanced radiation sensitivity for narrow-channel NMOS transistors. The radiation-induced threshold shift increases rapidly as the channel width decreased below 4 ?m. For example, with 4.5 ?m channel lengths the radiation sensitivity for 0.8 ?m wide FETs is twice that for 5 ?m wide devices. This geometry dependence can be explained qualitatively by two-dimensional potential calculations. These calculations show the fringing field influence on threshold voltage is reduced after radiation, leading to a larger total shift for the narrow channel devices.

Temperature-sensitive mutant of Schizosaccharomyces pombe exhibiting enhanced radiation sensitivity

A conditional lethal and radiation-sensitive mutant of Schizosaccharomyces pombe is described in which both characteristics result from a single gene mutation. Confirmation of the pleiotropic nature of this mutant was obtained by tetrad analysis and by testing the radiation sensitivity of a large number of revertants that grew normally at the restrictive temperature. The colony-forming ability of the mutant after ultraviolet radiation, gamma radiation, and ethyl methane sulfonate treatment is considerably altered by the post-treatment incubation temperature, showing higher survival at 25 than at 30degreesC. The radiosensitivity of the mutant is also influenced by the stage of growth. The difference in radiation sensitivity between the wild type and mutant is greater when log-phase cultures are compared. The characteristics of this mutant suggest that it is defective in a step common to both deoxyribonucleic acid replication and repair.

Radiation and hyperthermal response of normal tissue in situ.

Experiments were conducted to determine the effects of hyperthermal treatment on the radiation sensitivity of normal mouse skin. Hyperthermal treatment by itself produces no measurable response but enhances radiation sensitivity which increases with treatment temperature. Skin treated at 41° C was 1.18 times more sensitive than that treated at “normal” 37.5° C, and at 43° C the thermal enhancement ratio (TER) was 2.06. When compared with parallel studies using C3H mammary tumors, TER of 4.33 at 43° C, it appears that thermal sensitization of normal skin is appreciably less than for tumor. Hyperthermal treatment produces a favorable therapeutic ratio which increases with treatment temperature over the range studied.

Defective mammalian cells isolated from X-irradiated cultures

Cultures of mammalian cells exposed to ionizing radiations give rise to a large proportion of surviving cells which express radiation injury in an heritable manner, in that isolated progency of irradiated cells that give rise to abnormally small colonies are found to grow more slowly and are more sensitive to further irradiation. Such inherited injury occurs in irradiated human as well as hamster cell lines. In two defective cell lines investigated, the enhanced radiation sensitivity was accompanied by reduced oxygen consumption. Defective Chinese hamster cell lines can exist with no visible gross alterations in karotype. The frequency of small colony appearance in irradiated normal cultures increases regularly with dose, and the frequency with which they are produced is apparently too great to be due to single-gene mutations.

Nitric Oxide as a Modifier of Radiation Effects on Shigella Flexneri

SummaryThe mode of action of NO as a modifier of radiation sensitivity in aqueous suspensions of Shigella flexneri is different from that of O2.(1) NO present during irradiation has a sensitizing effect which is similar, but not chemically equivalent, to that due to O2.(2) Exposure to NO followed by removal of the gas prior to irradiation (i.e. pre-treatment) causes a latent irreversible effect on the bacteria, which becomes manifest after irradiation as enhanced radiation sensitivity dependent on concentration of NO and on the time of contact.(3) NO has also a relative protective effect.A tentative hypothesis based on the fixation of NO by unknown cell constituents is suggested.