Method for Measuring Breast Epithelial Turnover in Humans

Institution: University of California, Berkeley
Investigator(s): Marc Hellerstein, M.D., Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6JB-0139 Award: $197,000
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2000)
Altered division (proliferation) rates of cells is a central characteristic of all established cancers, including breast cancer. A remarkable, but well-documented feature about cancer is that the process of carcinogenesis itself (i.e., the sequence by which normal cells become cancerous) involves proliferation of cells.

Each time a cell divides, the likelihood of genetic mutation increases, and the risk for subsequent transformation to cancer increases cumulatively. Indeed, most of the breast cancer preventative interventions that have been proposed, such as tamoxifen treatment, are believed to work by reducing the proliferation rate of pre-cancerous cells and thereby slowing or preventing the development of cancer. It is therefore of obvious importance to have a reliable way for measuring the division rate of cells in tissues such as the breast, if we are to understand how cancer develops or test how cancer preventative agents truly work. Unfortunately, techniques for measuring cell proliferation have, until recently, either been unreliable or too toxic for use in human beings (due to the incorporation of radioactivity or mutagenic agents into DNA).

It is our belief that the field of breast cancer research has been hindered considerably by the absence of reliable cell division measurements applicable in people. In 1998, our laboratory presented a new technique for measuring cell division rates directly, accurately and safely in people, without use of radioactivity or toxic metabolites (Proceedings of the National Academy of Sciences, USA, 1998). This technique has been used to study the proliferation rates of T lymphocytes in AIDS patients (Nature Medicine, 1999) as well as several other cell types. We have recently worked out how to apply this technique to breast epithelial cells (the cells that become breast cancer) in animals.

The objectives of the present study are three-fold: first, to demonstrate that the technique is valid and can be applied in humans as well as animals; second, to determine what genistein, a potent cancer preventative agent in rats (found in soybeans) does to breast cell proliferation; and, third, to establish normal rates of breast cell division in humans and the factors that are associated with variability between women (e.g. age, weight, ethnicity, diet). Accordingly, we propose animal studies and human studies in this project. The animal studies will attempt to learn what effect genistein has on breast cell division; the human studies will attempt to learn the normal cell division rates and the nature of inter-individual variability. Our hope is that these exploratory, pilot studies will the groundwork for future large-scale trials assessing the role of breast cell proliferation carcinogenesis.


Final Report (2002)
Altered division (proliferation) rates of cells is a central characteristic of all established cancers, including breast cancer. Each time a cell divides, the likelihood of genetic mutation increases, and the risk for subsequent transformation to cancer increases. Indeed, most of the breast cancer preventative interventions that have been proposed, such as tamoxifen treatment, are believed to work by reducing the proliferation rate of pre-cancerous cells and thereby slowing or preventing the development of cancer. It is therefore of obvious importance to have a reliable way for measuring the division rate of cells in tissues such as the breast, if we are to understand how cancer develops or test how cancer preventative agents work.

The objectives of the present study are three-fold: first, to demonstrate that the technique is valid and can be applied in humans as well as animals; second, to determine the effects of genistein, a potent cancer preventative agent in rats (found in soybeans) on breast cell proliferation; and, third, to establish normal rates of breast cell division in humans and the factors that are associated with variability between women (e.g. age, weight, ethnicity, diet).

Thus far we have determined that our method can successfully measure proliferation in both animals and humans, and that routine clinical core biopsy methods are sufficient to measure breast cell proliferation in women. We have also determined that genistein significantly decreases breast cell proliferation, by 28%, in rats exposed to genistein both during pre-pubertal and adult life compared to those never exposed, and that there was a tendency for decreased cell proliferation if exposure was only during the pre-pubertal or adult stages, compared to those never exposed. It also seems that pre-pubertal exposure may in fact be most protective, however this will not be able to be established until more data is available.

We do not yet have sufficient data in human subjects to warrant analysis of variability in cell proliferation due to factors such as age, ethnicity, diet, etc. Since we can now apply this method in normal women via core biopsies we have begun to recruit more women in our study to allow future analyses of variability.

Our future goal is to determine if this newly developed method of measuring in vivo proliferation will effectively predict breast cancer occurrence and measure risk. To actualize this goal we will be looking to form a collaboration with an ongoing clinical study.


Symposium Abstract (2003)
We have developed a test for measuring breast (mammary) epithelial cell (MEC) proliferation in women. Briefly, this method entails having animals or human subjects drink a small amount of deuterated (2H2O) drinking water. DNA of breast cells that divide while this special water is in the body are labeled with deuterium (2H), then the amount of 2H incorporated, and thus percentage of new cells present, is determined.

The method was initially developed and validated in animal models. We demonstrated the sensitivity of the method for measuring changes in MEC proliferation due to pregnancy in mice and hormone treatment in rats with their ovaries removed (ovariecto-mized). Normal kinetics (rates) of MEC proliferation in rats and mice were also characterized. We concluded that extensive proliferation occurs in the pregnant mouse model, estrogen causes a dose-dependent increase in MEC proliferation in the ovariectomized rat, and that there appears to be both fast and slow turnover MEC populations within the mammary glands of rodents.

The method was then applied to a chemoprevention (cancer prevention) animal model. This study was designed to measure the effects of exposure to genistein (a possible cancer preventive agent) in pre-pubertal and adult rats, the interaction of genistein and estrogen in ovariectomized rats, and the effects of long-term dietary genistein exposure in ovariectomized rats. We conclude that exposure during both pre-pubertal and adult stages significantly suppressed MEC proliferation, but that exposure during either period by itself did not have significant effects. We also conclude that genistein did not have significant direct suppressive effects in ovariectomized adult rats treated with estrogen compared to controls, nor in adult ovariectomized rats fed a genistein containing diet for 13 weeks.

The method was then further developed for use in humans. We present here normative MEC proliferation data for women across a wide range of ages and varying menopausal status. We conclude that our method was successful in measuring MEC proliferation in women, that there is significant inter-individual and intra-individual variability in proliferation rates, and that both age and hormonal status significantly influence MEC proliferation rates in women. This method may be useful for measuring the effects of chemopreventive agents in women, upon further validation.