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.

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.

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.

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.

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.

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.

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.

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.

Production performance of white Leghorn layers limited fed.

Three trials were conducted to determine if the age at start of limited feeding or the amount of time laying hens were given to consume feed could be used to reduce feed intake without affecting egg weight (EW) or egg production. The age at start of limiting feed did not affect hen-day egg production (HDEP) or EW in any trial. In Trial 2, a significant difference in HDEP due to feeding times at 26 weeks of age was observed. Laying hens fed 7 hr/day at 26 weeks of age had a significantly lower HDEP than any other feeding time. Limited feeding in Trials 1 and 2 significantly decreased feed consumption, body weight gain (WG), and EW when compared with laying hens fed ad libitum. No difference in HDEP or EW was observed due to limited feeding in Trial 3. Laying hens fed 9 or 10 hr/day gained significantly less weight than laying hens fed ad libitum (Trial 3). Limited feeding did not improve feed efficiency in any trial. No consistent improvement in mortality could be attributed to limited feeding.

The effect of constant ambient temperature and ration on the performance of sexed broilers.

Two trials involving 480 Cobb color-sexed broiler chicks were conducted to determine the effect of various constant ambient temperatures on the performance of broilers. Temperatures in Trial 1 were 18 and 29 C and in Trial 2 were 24 and 35 C. The interacting effect of dietary energy (3.142 or 3.252 kcal ME/g of diet) and protein (16, 19, or 22%) on performance criteria was also examined within each trial. There was no indication of selective consumption of any of the ratios at any temperature. Differences in feed consumption observed in either trial were totally contributed by temperature effect. Within a trial, and irrespective of temperature treatment, the rate of growth and feed consumption of the females were less than that of the males. Males and females responded equally to the ambient temperature; there was no significant sex X temperature interaction in Trials 1 or 2.

Influence of toe-clipping and stocking density on laying hen performance.

Three experiments were conducted to investigate the influence of toe-clipping and bird density on laying hen performance. Toe-clipping was done on day-old chicks by removal of the digital claws from the front toes. Toe-clipped (TC) and intact (IN) pullets were assigned randomly to laying cages (Experiments 1 and 2, 19 weeks of age) or housed in similar body weight groups (Experiment 3, 18 weeks of age) at caging densities of either 4 (465 cm2/hen) or 5 (372 cm2/hen) hens per cage. Experiment 3 body weight groups were: heavy (greater than or equal to 1475 g), medium (greater than or equal to 1375 g, but less than 1475 g), light (greater than or equal to 1275 g, but less than 1375 g), and extra light (less than 1275 g). Body weights were determined at various ages during the grow-out and egg-laying periods. Beginning at 22 weeks of age, average daily egg weight, feed consumption, feed conversion, hen-day egg production, and mortality measures were made for 12 periods of lay of 28 days each. In Experiments 1 and 2, IN pullets were consistently heavier throughout the grow-out period and consumed significantly more feed during the egg laying period than TC birds. Significantly greater average daily egg weights were found in IN than in TC hens in Experiment 1 but not in Experiment 2. Increasing the number of hens from 4 to 5 hens per cage resulted in a significant reduction in feed intake and body weight gain in Experiments 1, 2, and 3. In Experiment 1, mean daily egg weight was significantly increased (.11 g) upon crowding. In Experiment 2, crowding elevated mortality. In Experiments 1 and 2, but not 3, a significant toe treatment by bird density interaction was observed for hen-day egg production. The IN birds had lowered hen-day egg production rates when crowded than when they were afforded more space, whereas hen-day egg production was elevated in crowded TC hens when compared to TC hens housed at the less crowded density. In Experiment 3, an initial (4 weeks of age) significant depression in pullet body weight was found in the TC pullets but disappeared by the 8th week. Feed usage was also significantly greater in IN than in TC hens in Experiment 3. Toe treatment did not affect any other hen performance variable measured. Egg weight, feed intake, and feed conversion measures varied by body weight groups. In general, the heavier hens consumed more feed and laid heavier eggs, but they were less efficient in converting feed into eggs.

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.

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.

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.