Time-dose relationships in radiation-enhanced integration.

PURPOSE: We have shown that ionizing radiation increases recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. This paper reports the duration of increased recombination after irradiation. MATERIALS AND METHODS: A549 or NIH/3T3 cells were transfected at various times after irradiation. Cells were also irradiated with several fractionation schemes and then transfected. RESULTS: Enhanced integration (EI) is a very long-lived process, lasting at least 2-3 days after single radiation fractions. The duration of EI activation is radiation dose-dependent. The efficiency of EI is dependent on radiation dose and independent of fractionation, such that low dose-rate, fractionated and single radiation doses result in similar levels of EI when corrected for differences in cytotoxicity. CONCLUSIONS: Radiation, given with fraction sizes and dose-rates used in clinical radiation therapy, induces a long-lived hyper-recombination state. Since radiotherapy is already a component of treatment for many malignancies and is integrated into radiation-gene therapy trials, an understanding of recombination events that improve gene delivery is important and timely.

RhoB is required to mediate apoptosis in neoplastically transformed cells after DNA damage.

The effect of neoplastic transformation on the response to genotoxic stress is of significant clinical interest. In this study, we offer genetic evidence that the apoptotic response of neoplastically transformed cells to DNA damage requires RhoB, a member of the Rho family of actin cytoskeletal regulators. Targeted deletion of the rhoB gene did not affect cell cycle arrest in either normal or transformed cells after exposure to doxorubicin or gamma irradiation, but rendered transformed cells resistant to apoptosis. This effect was specific insofar as rhoB deletion did not affect apoptotic susceptibility to agents that do not damage DNA. However, rhoB deletion also affected apoptotic susceptibility to Taxol, an agent that disrupts microtubule dynamics. We have demonstrated that RhoB alteration mediates the proapoptotic and antineoplastic effects of farnesyltransferase inhibitors, and we show here that RhoB alteration is also crucial for farnesyltransferase inhibitors to sensitize neoplastic cells to DNA damage-induced cell death. We found RhoB to be an important determinant of long-term survival in vitro and tumor response in vivo after gamma irradiation. Our findings identify a pivotal role for RhoB in the apoptotic response of neoplastic cells to DNA damage at a novel regulatory point that may involve the actin cytoskeleton.

The Ras radiation resistance pathway.

The critical pathways determining the resistance of tumor cells to ionizing radiation are poorly defined. Because the ras oncogene, a gene activated in many human cancers treated with radiotherapy, can induce increased radioresistance, we have asked which Ras effector pathways are significant in conferring a survival advantage to tumor cells. The phosphoinositide-3-kinase (PI3K) inhibitor LY294002 radiosensitized cells bearing mutant ras oncogenes, but the survival of cells with wild-type ras was not affected. Inhibition of the PI3K downstream target p70S6K by rapamycin, the Raf-MEK-MAPK pathway with PD98059, or the Ras-MEK kinase-p38 pathway with SB203580 had no effect on radiation survival in cells with oncogenic ras. Expression of active PI3K in cells with wild-type ras resulted in increased radiation resistance that could be inhibited by LY294002. These experiments have indicated the importance of PI3K in mediating enhanced radioresistance and have implicated PI3K as a potential target for specific radiosensitization of tumors.

Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines.

Transformation with ras oncogenes results in increased radiation sur vival in many but not all cells. In addition, prenyltransferase inhibitors which inhibit ras proteins by blocking posttranslational modification radiosensitize cells with oncogenic ras. These findings suggest that oncogenic ras contributes to intrinsic radiation resistance. However, because introduction of ras oncogenes does not increase radiation survival in all cells and because prenyltransferase inhibitors target molecules other than ras, these studies left the conclusion that ras increases the intrinsic radi ation resistance of tumor cells in doubt. Here we show that genetic inactivation of K- or N-ras oncogenes in human tumor cells (DLD-1 and HT1080, respectively) leads to increased radiosensitivity. Reintroduction of the activated N-ras gene into the HT1080 line, having lost its mutant allele, resulted in increased radiation resistance. This study lends further support to the hypothesis that expression of activated ras can contribute to intrinsic radiation resistance in human tumor cells and extends this finding to the K- and N- members of the ras family. These findings support the development of strategies that target ras for inactivation in the treatment of cancer.

Farnesyltransferase inhibitors potentiate the antitumor effect of radiation on a human tumor xenograft expressing activated HRAS.

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.

Radiation-induced recombination is dependent on Ku80.

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.

DNA damaging agents improve stable gene transfer efficiency in mammalian cells.

Gene therapy is an evolving discipline which today relies primarily on viral systems for gene transfer. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that both ionizing irradiation and hydrogen peroxide can each increase the gene transfer efficiency of plasmids. Hydrogen peroxide improves gene transfer in a linear dose-dependent manner. At equitoxic doses, hydrogen peroxide improves gene transfer by 20-fold over untreated cells and approximately 5 times above that seen for radiation, and this improvement correlates with both the total amount of DNA damage induced and the amount of residual damage after 4 hr of repair. These data suggest that DNA damaging agents may be useful to improve human gene therapy.

High-efficiency stable gene transfer of adenovirus into mammalian cells using ionizing radiation.

We report a novel method for targeting adenovirus-mediated gene delivery. By irradiating mammalian cells prior to adenoviral transduction, adenoviral gene transfer is greatly improved and the adenoviral genome integrates into cellular DNA. In this work, human and rodent cell lines were irradiated and subsequently transduced with the adenovirus vector Ad5CMVlacZ. Initial levels of transduction were as much as 40-fold higher in irradiated cells, and this improvement in transduction was radiation dose dependent. The duration of lacZ expression in irradiated cells was also much longer than in nonirradiated cells and reached a plateau after 21 days. At doses of 7 Gy, long-term (< 50 day) expression of lacZ could be detected in 15% of cells by flow cytometry. This long-lasting expression of lacZ was due to viral DNA integration into the host genome. Thus, pretreatment of cells with ionizing radiation improves both immediate transduction efficiency and duration of transgene expression. This may lead to the development of new protocols combining radiation and gene therapy in treating human malignancy.

Effect of oncogene transformation of rat embryo cells on cellular oxygen consumption and glycolysis.

We found an unique effect of oncogene transfections on rat embryo cell (REF) respiration, glycolysis and radiation response. Radioresistance, defined as an increase in Do, increases for REF cells transfected with v-myc or H-ras oncogenes. The combination of both oncogenes confers the maximal radioresistance. Our work shows inhibition of oxygen uptake when cells are transfected with v-myc or H-ras alone. However, oxygen uptake increases when cells are transfected simultaneously with v-myc + H-ras (3.7,2.1,2.8). A higher oxygen consumption results from increased utilization of pyruvate via the Kreb's cycle. Succinate stimulates cellular oxygen consumption. The maximum stimulation of oxygen consumption by succinate occurred with v-myc + H-ras transfected cells. The glycolysis of the transfected cells is also altered by the oncogenes. Our glycolytic measurements indicate the H-ras oncogene causes the largest stimulation of glycolysis. Our data shows that transfection with oncogenes has a major effect on cellular glycolysis, oxidative metabolism as well as the subsequent radiation response.

Intravascular oxygen distribution in subcutaneous 9L tumors and radiation sensitivity.

Phosphorescence quenching was evaluated as a technique for measuring PO2 in tumors and for determining the effect of increased PO2 on sensitivity of the tumors to radiation. Suspensions of cultured 9L cells or small pieces of solid tumors from 9L cells were injected subcutaneously on the hindquarter of rats, and tumors were grown to between 0.2 and 1.0 cm in diameter. Oxygen-dependent quenching of the phosphorescence of intravenously injected Pd-meso-tetra-(4-carboxyphenyl) porphine was used to image the in vivo distribution of PO2 in the vasculature of small tumors and surrounding tissue. Maps (512 x 480 pixels) of tissue oxygen distribution showed that the PO2 within 9L tumors was low (2-12 Torr) relative to the surrounding muscle tissue (20-40 Torr). When the rats were given 100% oxygen or carbogen (95% O2-5% CO2) to breathe, the PO2 in the tumors increased significantly. This increase was variable among tumors and was greater with carbogen compared with 100% oxygen. Based on irradiation and regrowth studies, carbogen breathing increased the sensitivity of the tumors to radiation. This is consistent with the measured increase in PO2 in the tumor vasculature. It is concluded that phosphorescence quenching can be used for noninvasive determination of the oxygenation of tumors. This method for oxygen measurements has great potential for clinical application in tumor identification and therapy.

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.

Instability of hydrogen peroxide solutions was noted during the experimental exposure of human cells in culture to hydrogen peroxide in experiments designed to study the production and repair of DNA single-strand breaks. A hydrogen peroxide concentrate was diluted into culture medium, which was then added to experimental cell cultures at various times, with all cultures assessed for DNA damage at 2 h. Only cells treated by the first addition had observable DNA damage. This result was unexpected since these cells had had the maximum repair time. It was determined that the hydrogen peroxide had been eliminated by the culture medium. To determine the mechanism of this elimination, 200 microM hydrogen peroxide was added to various cell culture components, and the solutions were assayed for hydrogen peroxide after 1 h at 37 degrees C. Although most components (except the balanced salts) showed some hydrogen peroxide degradation, it was found that sodium pyruvate was most effective, by a wide margin, in eliminating hydrogen peroxide and its toxic effects. This was confirmed by addition of pyruvate to balanced salt solutions or buffers, and observing the same elimination of hydrogen peroxide. We subsequently found a few earlier reports describing the decarboxylation reaction between hydrogen peroxide and pyruvate, but no kinetic measurements have been published and there seems to be no general appreciation for the very high efficiency of this reaction. The present work presents a preliminary assessment of the importance of pyruvate in the study of hydrogen peroxide and other reactive oxygen species in mammalian cell culture.

Ionizing radiation greatly improves gene transfer efficiency in mammalian cells.

The vast majority of clinical protocols involving gene therapy today rely on viral vectors for gene transduction. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that ionizing radiation can improve plasmid transfection efficiency in both normal and neoplastic human and mouse cells. As high as 1,400-fold improvement in transfection efficiency can be seen in primary human fibroblasts treated with 9 Gy. Radiation improves transfection efficiency in a dose-dependent manner of only linearized plasmid DNA in transformed or immortalized cells, but of both linearized and supercoiled plasmid in normal human fibroblasts. The gene transfer dose-response curves are linear for neoplastic cell lines and exponential for primary cell lines. This suggests that radiation can improve gene integration by at least two mechanisms, one that may require free DNA ends and one that does not. The 2-hr delay described here, from the time of irradiation to the beginning of enhanced gene integration, suggests an inducible process that becomes active after the bulk of the radiation damage has been repaired. Our data further suggest that radiation may be useful to target human gene therapy using plasmid vectors.

Intracellular accumulation of ofloxacin-loaded liposomes in human synovial fibroblasts.

In order to incorporate ofloxacin within liposomes, the reverse-phase evaporation technique was carried out. The liposome lipid matrix consisted of dipalmitoylphosphatidylcholine-cholesterol-dihexadecylphosphate (4: 3:4 molar ratio). The liposome formulation presented a mean size of 185 +/- 31 nm and had an encapsulation capacity of 5.3 microliters/mumol. The liposome formulation was able to deliver ofloxacin into McCoy cells in a greater amount (2.6-fold) than the free drug, improving antibiotic accumulation.

In-vitro antimycoplasmal activity of flurithromycin.

The in vitro activity of flurithromycin, a 14-membered macrolide drug, was found to be similar to that of erythromycin against 41 strains of Mycoplasma spp. and 100 strains of Ureaplasma urealyticum. All 28 strains of Mycoplasma hominis were uniformly resistant to both macrolides with MICs > 256 mg/L, U. urealyticum showed intermediate resistance with MIC50s of 0.5 and 1 mg/L for erythromycin and flurithromycin, respectively, whereas the ten strains of Mycoplasma pneumonia were susceptible to < or = 0.03 mg/L of both macrolides.

In vitro antimycoplasmal activities of rufloxacin and its metabolite MF 922.

The in vitro activities of rufloxacin and its metabolite, MF 922, were compared with those of ofloxacin, ciprofloxacin, erythromycin, and minocycline against Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma fermentans, and Ureaplasma urealyticum. Rufloxacin, MF 922, and ciprofloxacin shared similar activities against all mycoplasmas tested. (MICs for 90% of isolates tested [MIC90s], 0.5 to 4 micrograms/ml. Ofloxacin had the lowest MIC90s for U. urealyticum, M. fermentans, and M. hominis (MIC90s, 0.25 to 1 micrograms/ml) and erythromycin had the lowest MIC90 for M. pneumoniae (MIC90, 0.004 micrograms/ml).

Localization of tumors and evaluation of their state of oxygenation by phosphorescence imaging.

Oxygen-dependent quenching of phosphorescence has been used to image the distribution of oxygen pressure in small tumors and surrounding tissue. Suspensions of cultured 9L cells or small pieces of solid tumors from 9L cells were injected into the surface of the muscle of the hindquarter of rats, and the tumors were grown until they were 0.2-1.0 cm in diameter. The phosphorescent probe for oxygen was injected into the systemic blood, and phosphorescence was imaged with a video camera. Images of the phosphorescence were collected using a series of different delay times after illumination with a light flash (less than 5-microseconds width at half-height), and the phosphorescence decay constants (lifetimes) and oxygen pressure were calculated for each pixel of the image arrays. The areas of tissue within the tumors were observed to have increased phosphorescence lifetimes and lower oxygen pressures than the surrounding tissue. Phosphorescence imaging is, therefore, a noninvasive optical method which permits quantitation of the distribution of oxygen in small tumors and also, at least in the 9L tumors, differentiation of tumor from normal tissue.

Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples.

By combining the least complicated and expedient methods of sample handling with the sensitivity and specificity of the GSH assay by enzymatic recycling and the small volumes and software capabilities of microtiter plate technology we have devised a rapid, sensitive, and easy assay for GSH and GSSG in biological samples. The assay is sensitive to 5 pmol in sample volumes of 50 microliters, although other volumes could be used. The use of a computer-driven microplate with software capable of linear kinetic data storage and analysis on each well, Maxline series microplate readers and Softmax software, enables the user not only to assay large numbers of samples per day but also to have immediate calculated results. We suggest by examples that measurements of total GSH as well as changes in GSH:GSSG in vitro and in vivo are feasible with this technology.

Production performance of White Leghorn layers fed lactobacillus fermentation products.

A series of trials was conducted to determine if adding a Lactobacillus fermentation product (LAC) to the feed of laying hens would improve their production performance. Feeding a liquid, nonviable LAC product to either cage or floor housed laying hens did not improve hen-day egg production, feed efficiency, nor egg size during a 48 week experimental period. Laying hens fed a dried, nonviable LAC product did not show any improvement in hen-day egg production nor feed efficiency compared with laying hens fed no LAC or zinc bacitracin. Addition of a viable LAC product to ratios of differing protein levels did not improve hen-day egg production, livability, or egg size of laying hens.