Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide.

Previously, we demonstrated that A549, a human lung cancer cell line, could be adapted to the free radical nitric oxide (NO●). NO● is known to be over expressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serve as a useful model system to study the biology of NO●. To see if tumor cells can similarly be adapted to any free radical with the same outcome, herein we successfully adapted A549 cells to high levels of hydrogen peroxide (HHP). A549-HHP, the resulting cell line, was more resistant and grew better then the parent cell line, and showed the following characteristics: (1) resistance to hydrogen peroxide, (2) resistance to NO●, (3) growth with and without hydrogen peroxide, and (4) resistance to doxorubicin. Gene chip analysis was used to determine the global gene expression changes between A549-parent and A549-HHP and revealed significant changes in the expression of over 1,700 genes. This gene profile was markedly different from that obtained from the A549-HNO cell line. The mitochondrial DNA content of the A549-HHP line determined by quantitative PCR favored a change for a more anaerobic metabolic profile. Our findings suggest that any free radical can induce resistance to other free radicals; this is especially important given that radiation therapy and many chemotherapeutic agents exert their effect via free radicals. Utilizing this model system to better understand the role of free radicals in tumor biology will help to develop new therapeutic approaches to treat lung cancer.

Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines.

The free radical nitric oxide (NO) is over-expressed in many tumors, including head and neck squamous cell carcinomas (HNSCC); however, the role NO plays in tumor pathophysiology is still not well understood. We, herein, report the development of an in vitro model system which can be used to probe the role of NO in the carcinogenesis of HNSCC. Five HNSCC cell lines were adapted to a high NO (HNO) environment by gradually introducing increasing concentrations of DETA-NONOate, a nitrogen-based NO donor, to cell media. The adaptation process was carried out until a sufficiently high enough donor concentration was reached which enabled the HNO cells to survive and grow, but which was lethal to the original, unadapted ("parent") cells. The adapted HNO cells exhibited analogous morphology to the parent cells, but grew better than their corresponding parent cells in normal media, on soft agar, and in the presence of hydrogen peroxide, an oxygen-based free radical donor. These results indicate that the HNO cell lines are unique and possess biologically different properties than the parent cell lines from which they originated. The HNO/parent cell lines developed herein may be used as a model system to better understand the role NO plays in HNSCC carcinogenesis.

Nitric oxide: perspectives and emerging studies of a well known cytotoxin.

The free radical nitric oxide (NO(*)) is known to play a dual role in human physiology and pathophysiology. At low levels, NO(*) can protect cells; however, at higher levels, NO(*) is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO(*) in cancer has to date been tissue-specific, we herein review underlying commonalities of NO(*) which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO(*) in human physiology and pathophysiology, including the very important relationship between NO(*) and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO(*) in three main areas of epigenetics-DNA methylation, microRNAs, and histone modifications-is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO(*) (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO(*) in cancer.

Photodynamic therapy: occupational hazards and preventative recommendations for clinical administration by healthcare providers.

OBJECTIVE: Photodynamic therapy (PDT) as a medical treatment for cancers is an increasing practice in clinical settings, as new photosensitizing chemicals and light source technologies are developed and applied. PDT involves dosing patients with photosensitizing drugs, and then exposing them to light using a directed energy device in order to manifest a therapeutic effect. Healthcare professionals providing PDT should be aware of potential occupational health and safety hazards posed by these treatment devices and photosensitizing agents administered to patients. MATERIALS AND METHODS: Here we outline and identify pertinent health and safety considerations to be taken by healthcare staff during PDT procedures. RESULTS: Physical hazards (for example, non-ionizing radiation generated by the light-emitting device, with potential for skin and eye exposure) and chemical hazards (including the photosensitizing agents administered to patients that have the potential for exposure via skin, subcutaneous, ingestion, or inhalation routes) must be considered for safe use of PDT by the healthcare professional. CONCLUSIONS: Engineering, administrative, and personal protective equipment controls are recommendations for the safe use and handling of PDT agents and light-emitting technologies.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.