Investigation of the effects of oxidative stress on mouse keratinocytes in culture and the relationship between reactive oxygen species and tumor promotion

• Main Author: Przybyszewski, Joseph
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1996
• Subjects: Biophysics; Cellular biology; Oncology
• ISBN: 9798684662379

A considerable amount of circumstantial evidence exists supporting the role for reactive oxygen species (ROS) in tumor promotion; however, the mechanism for this relationship is unknown. In order to investigate the role of oxidative stress in tumor promotion, the studies of this dissertation are divided into two sections. First, induction of oxidative DNA damage, in the form of 8-hydroxydeoxyguanosine (8OHdG), is established in oxidative-stressed mouse keratinocytes in culture using high performance liquid chromatography with electrochemical detection (HPLC-EC). Second, measurements of ROS (by luminol-dependent chemiluminescence (LDCL)) and 8OHdG in DNA (by HPLC-EC) of keratinocytes treated with the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), are used to investigate the role of oxidative stress in tumor promotion in vitro. Because most studies of phorbol ester-mediated tumor promotion have examined oxidative stress in vivo or in co-cultures of phagocytic cells with non-phagocytes, the investigation of oxidative stress in a cultured keratinocyte tumor promotion model system is warranted. In the first section, the reliability and consistency of measurements of oxidative stress in keratinocytes in vitro was demonstrated by (1) the narrow range of background 8OHdG values and the reproducibility of trends in 8OHdG formation, (2) two-fold increases in 8OHdG formation after treatment of cells with 600 J/m$\sp{2}$ UVC or 630 mJ/cm$\sp{2}$ UVB, (3) 6-fold and 4-fold increases in 8OHdG formation after treatment of cells with 5 mM $\rm H\sb{2}O\sb{2}$ in combination with 360 J/m$\sp{2}$ UVC and 630 mJ/cm$\sp{2}$ UVB, respectively, and (4) a significant and comparable increase in UVB-induced formation of the formamido remnant of thymine, also a measure of oxidative stress and seen for the first time in DNA of cells. In the second section, the sensitivity of LDCL in measuring ROS produced by chemical agents or by cells exogenously or endogenously is demonstrated. This sensitivity permitted detection of a TPA-induced increase in ROS levels which were too low to induce 8OHdG formation (even under a variety of treatment conditions) in DNA of keratinocytes. Thus, compared to the high levels of exogenous ROS produced in vivo by TPA-activated phagocytes, low levels of ROS generated by the keratinocytes themselves do not damage DNA but may be involved in signal transduction pathways.