Role of Oxidative DNA Damage to Breast Tumor Progression

Institution: Lawrence Livermore National Laboratory
Investigator(s): Paul Henderson, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9KB-0179 Award: $588,555
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2003)
A classic theory of carcinogenesis proposes that DNA is damaged by environmental factors, which lead to mutations in the DNA code. Once enough mutations accumulate, cells can no longer function properly, and cancer arises. The facts that smoking causes lung cancer, and that sunlight exposure causes skin cancer support this theory. However, such a direct environmental link is not evident for breast cancer in humans. A more contemporary theory suggests that-the metabolism of oxygen produces free radicals, such as hydrogen peroxide, and these cause carcinogenic DNA damage. In order to validate the latter theory, it is necessary to measure the products of free radical damage in cells. Damage to cells caused by reactive oxygen species is called oxidation.

In some breast cancers, increased levels of oxidative DNA damage have been associated with tumor progression. However, the most commonly used marker of DNA oxidation, called 8-oxoG, is known to be chemically unstable, and is difficult to measure accurately. Importantly, 8-oxoG is itself easily oxidized to form several secondary oxidation products. Recently, I have determined that these secondary products are chemically stable, and that they are highly mutagenic. However, the products have not been studied in breast cancer, since they are difficult to detect with current technology. Thus, I propose to develop new technology that will allow detection of the 8-oxoG oxidation products in rat and human breast cancer cells.

We will chemically synthesize a radioactively labeled DNA building block, which can be tested in model cell and animal models as a “surrogate marker” to measure oxidative DNA damage. The radioactive label is easy to detect, and will allow very low concentrations of DNA damage to be assayed. The building block will be introduced to breast cancer cells, which will incorporate it into new DNA during tumor growth. We expect that the radioactive building block will be damaged by free radicals inside the cancer cell, and that the DNA damage products will contribute to the progression of the cancer. After isolation of DNA from the cells, the radioactive label will be used to determine how much of the building block has been incorporated into the cell, and to what extent the DNA of the cell is damaged. The technology will be developed using human breast cancer cells grown in cell culture, which are easy to handle. Then, as a model of human disease, breast cancer-prone rats will be fed the building block. At different times during breast cancer development in the rats, their breast tissue will be isolated and tested for oxidized DNA damage. The stable, radioactively-labeled biomarkers can be detected at remarkably low levels, which will allow the study of the typical, “real life” levels of DNA damage. Lawrence Livermore National Laboratory is a world leader in biological applications of radiolabel detection.

Such a novel DNA oxidative damage-based assay can be used as a foundation for making diagnostics for use in future human clinical and epidemiological studies. An additional benefit of these biomarkers will be their eventual application to chemoprevention of cancer.

Final Report (2006)
The goal of the project was to study the role of several recently discovered DNA oxidation products in breast carcinogenesis. We believe that these products cause mutations, if left unrepaired. In addition, their concentration in cells may increase with tumor progression to initiate the mutations needed for advanced stages of breast cancer.

We initially began the project by synthesizing one of these products, called 8-oxodG, with a radiocarbon label in the molecule. This label allows the 8-oxodG to be detected using an accelerator mass spectrometer (AMS). The 8-oxodG was “feed” to cultures breast cancer cells and to mice bearing breast tumors. In the cell culture portion of the project we found that the 8-oxodG was incorporated into DNA as measured by AMS. We expected to see the compound get converted to several oxidation products, but this did not occur. In an effort to "force" the cells to oxidize 8-oxodG we dosed them with estradiol (E2), a hormone that is present in breast tissue that is known to be carcinogenic. Upon exposure to E2 the 8-oxodG in the DNA became damaged at concentrations of the hormone that are relevant to those present in human breast tissue (even in-post menopausal women). This observation supports a novel mechanism by which DNA can be damaged in a hormone-specific manner that may play a role in breast carcinogenesis. We believe that oxidative stress caused by the E2 oxidizes either the DNA or the nucleotides involved in DNA synthesis. The mechanistic aspects of this observation were further probed by measuring protein levels of a key repair enzyme, called MTH1 that targets 8-oxodG metabolites in cells. This repair protein was shown to increase in concentration with increasing E2 concentration. We also performed experiments to determine mechanism of 8-oxodG incorporation into DNA and RNA, which may have relevance to altered protein function. All of the related pathways are currently targets for anti-cancer drugs, and it may be desirable and possible to exclude 8-oxodG from being incorporated into cancer cells in order to slow the mutation rate of the cells.

In a mouse model of ductal carcinoma in situ we also observed incorporation of the 8-oxodG into tumor DNA. The AMS data indicate that most of the 8- oxodG was oxidized or converted to other species even in the absence of supplemental E2. The data indicate that in advanced stage tumors the oxidative stress present is sufficiently severe to oxidize the DNA spontaneously.

In conclusion, we have begun to gain a molecular-level understanding of the cancer initiation and progression with respect to mutagenic DNA damage. It may be possible to use the types and concentrations of these new DNA damage products as a "fingerprint" for cancer diagnosis or response to treatment.

Symposium Abstract (2005)
One increasingly studied source of cancer initiation and progression is the endogenous formation of mutagenic DNA damage from reactive oxygen species (ROS), which are produced by processes such as cellular respiration and the activation of macrophages. One of the major products of DNA oxidation is 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), a mutagenic adduct whose elevated levels in many cancerous tumors have been correlated to altered DNA repair capacity and increases in ROS production. It is, however, known that 8-oxodG is highly susceptible in vitro to oxidation by ROS such as singlet oxygen and hydroxy radicals to form a variety of mutagenic nucleobase derivatives. Detection of these products in vivo has been thwarted by insufficient sensitivity of traditional mass spectrometry methods, since the putative adducts are expected to be present at levels of 1 per 107-1010 normal nucleotides. In this study we developed a radiochemical accelerator mass spectrometry (AMS) method to detect 8- oxodG oxidation by tracing the fate of 14C-labeled 8-oxodG in MCF-7 human breast cancer cells.

When cultured MCF-7 cells were dosed with 14C-labeled 8-oxodG, incorporation of the 14C-label into DNA and subsequent oxidation was robustly monitored by AMS. The labeled nucleoside was found to be incorporated into DNA at levels of ~6000 adducts per cell, which is near the basal level of the lesion in normal cells, and 8-oxodG was partially oxidized to one of the expected products, a putative cyanuric acid derivative, at ~200 adducts per cell. Supplemental 17b-estradiol caused the formation of several yet to be characterized products, which we hypothesize to play a role in hormone-dependent cancer cell proliferation. Work was performed at the Research Resource for Biomedical AMS, operated at UC LLNL under the auspices of the U.S. DOE contract #W-7405-ENG-48 and partially supported by NIH/NCRR, Biomedical Technology Program grant #P41 RR13461.

Symposium Abstract (2005)
We report the formation of newly discovered mutagenic DNA damage products that form in cultured human breast cancer cells upon exposure to estrogen. A recently developed technology called accelerator mass spectrometry was used to detect the damage, which form at low but potentially relevant concentrations. Most of the experiments were performed with cultured human breast cancer cells, but the results are currently being reproduced in a mouse model of a type of breast cancer called ductal carcinoma in situ. Since the DNA damage products are mutagenic, we hypothesize that they cause modifications in the DNA sequence of estrogen responsive breast cells leading to the initiation and progression of breast cancer.

A sample preparation protocol for quantification of radiolabeled nucleoside incorporation into DNA by accelerator mass spectrometry
Index Medicus: Science
Authors: S Haha, J Mundta, E Ubicka, K Turteltauba, J Gregg, P Henderson
Yr: 2007 Vol: 259 Nbr: 1 Abs: Pg:763-766

Urea Lesion Formation in DNA as a Consequence of 7,8-Dihydro-8-oxoguanine Oxidation and Hydrolysis Provides a Potent Source of Point Mutations
Periodical:Chemical Research in Toxicology
Index Medicus: Chem Res Toxicol
Authors: Henderson PT, Neely WL, Delaney JC, Gu F, Niles JC, Hah SS, Tannenbaum SR, and Essigmann J
Yr: 2005 Vol: 18 Nbr: Abs: Pg:12-18

Hydantoin derivative formation from oxidation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and incorporation of 14C-labeled 8-oxodG into the DNA of human breast cancer cells.
Periodical:Bioorganic Medical Chemistry
Index Medicus: Bio Med Chem
Authors: Hah SS, Kim HM, Sumbad RA, Henderson PT.
Yr: 2005 Vol: Nbr: 15 Abs: Pg:3627-31

Measurement of 7,8-dihydro-8-oxo-2'-deoxyguanosine metabolism in MCF-7 cells at low concentrations using accelerator mass spectrometry.
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Hah SS, Mundt JM, Kim HM, Sumbad RA, Turteltaub KW, Henderson PT
Yr: 2007 Vol: 104 Nbr: 27 Abs: Pg:11203-8