Induction of reactive oxygen species without 8-hydroxydeoxyguanosine formation in DNA of initiated mouse keratinocytes treated with 12-O-tetradecanoylphorbol-13-acetate

Joseph Przybyszewski, Harold C. Box, Molly Kulesz-Martin

Research output: Contribution to journalArticle

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Abstract

Evidence for the involvement of oxidative stress in 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion has focused on non-initiated immune cells, tumor cell lines and non-initiated epidermis treated in vivo. This paper reports the effects of TPA on 8-hydroxydeoxyguanosine (8OHdG) formation and the generation of reactive oxygen species (ROS) in cloned initiated mouse epidermal keratinocytes in order to determine whether TPA can directly damage DNA through ROS production within the keratinocytes. Using high performance liquid chromatography with electrochemical detection (HPLC-EC). TPA did not induce 8OHdG formation in DNA of initiated keratinocytes treated under a variety of conditions. The reliability of the HPLC-EC system is demonstrated by (i) the linearity of the 8OHdG standard curve; (ii) the consistency of 8OHdG measurements in calf thymus and cellular DNA; and (iii) the dose-dependent increase in 8OHdG in DNA of initiated keratinocytes treated with UVC in the presence and absence of H2O2. Though not DNA-damaging, TPA induced a 65% increase in ROS (P <0.05) as detected by luminol-dependent chemiluminescence. These results support a mechanism for the role of oxidative stress in tumor promotion that does not involve direct DNA damage to the keratinocyte target cell. The relationship between ROS, signal transduction and tumor promotion is discussed in light of the above results which is consistent with the role of TPA-induced ROS as second messengers in signal transduction.

Original languageEnglish (US)
Pages (from-to)1467-1474
Number of pages8
JournalCarcinogenesis
Volume19
Issue number8
DOIs
StatePublished - Aug 1998
Externally publishedYes

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Tetradecanoylphorbol Acetate
Keratinocytes
Reactive Oxygen Species
DNA
DNA Damage
Signal Transduction
Oxidative Stress
High Pressure Liquid Chromatography
Luminol
Neoplasms
Second Messenger Systems
Luminescence
Tumor Cell Line
Epidermis
8-oxo-7-hydrodeoxyguanosine

ASJC Scopus subject areas

  • Cancer Research

Cite this

Induction of reactive oxygen species without 8-hydroxydeoxyguanosine formation in DNA of initiated mouse keratinocytes treated with 12-O-tetradecanoylphorbol-13-acetate. / Przybyszewski, Joseph; Box, Harold C.; Kulesz-Martin, Molly.

In: Carcinogenesis, Vol. 19, No. 8, 08.1998, p. 1467-1474.

Research output: Contribution to journalArticle

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abstract = "Evidence for the involvement of oxidative stress in 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion has focused on non-initiated immune cells, tumor cell lines and non-initiated epidermis treated in vivo. This paper reports the effects of TPA on 8-hydroxydeoxyguanosine (8OHdG) formation and the generation of reactive oxygen species (ROS) in cloned initiated mouse epidermal keratinocytes in order to determine whether TPA can directly damage DNA through ROS production within the keratinocytes. Using high performance liquid chromatography with electrochemical detection (HPLC-EC). TPA did not induce 8OHdG formation in DNA of initiated keratinocytes treated under a variety of conditions. The reliability of the HPLC-EC system is demonstrated by (i) the linearity of the 8OHdG standard curve; (ii) the consistency of 8OHdG measurements in calf thymus and cellular DNA; and (iii) the dose-dependent increase in 8OHdG in DNA of initiated keratinocytes treated with UVC in the presence and absence of H2O2. Though not DNA-damaging, TPA induced a 65{\%} increase in ROS (P <0.05) as detected by luminol-dependent chemiluminescence. These results support a mechanism for the role of oxidative stress in tumor promotion that does not involve direct DNA damage to the keratinocyte target cell. The relationship between ROS, signal transduction and tumor promotion is discussed in light of the above results which is consistent with the role of TPA-induced ROS as second messengers in signal transduction.",
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