The development of the mammary gland is under the control of the hypothalamic-pituitary-gonadal (HPG) axis. Hormones secreted by the ovary and the pituitary and the timing of cell division are in turn affected by circadian rhythms controlled by the master circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus whose 24-hour rhythm is entrained by light reaching the retina. The SCN controls both central and peripheral clock genes as well as the release of gonadotropin and luteinizing hormone releasing hormone (GnRH/LHRH) neurons. Because the stage of development and rate of cell proliferation in the mammary gland are essential for cancer initiation, we postulate that the susceptibility of this organ to be transformed by chemical carcinogens might be in great part regulated by circadian rhythms, and that disruption in circadian gene expression could conceivably result in deregulation of these processes and contribute to tumor development. This study was designed with the purpose of characterizing the pattern of gene expression in the rat mammary gland during a 24-hour circadian cycle under standard light conditions. For this purpose, 50 day-old female virgin Sprague-Dawley rats were maintained in a light controlled animal facility with a12 hour light (lights on at 8:00 hr-Zeitgeber [Zt] 0) /12 hour darkness cycle (lights off at 20:00 hr-Zt 12). After 2 weeks in this environment three animals were euthanized for the collection of mammary tissues at six time points during a 24 hr period, starting at 12:00 hr (Zt4), and continuing at 16:00 hr (Zt8), 20:00 hr (Zt12), 24:00 hr (Zt16), at 4:00 hr (Zt20), and at 8:00 hr (Zt24). Total RNA was isolated from frozen mammary glands using standard techniques. RNA quantity was measured by Nanodrop and RNA integrity was assessed by capillary electrophoresis RNA chip before its use for determining the level of expression by real time RT-PCR of the following genes: Period (Per) 1, 2, and 3, Cryptochrome (Cry) 1 and 2, Differentiated 1 or embryo chondrocytes 1 (bhlhb2 or Sharp 1), Differentiated 2 or embryo chondrocytes 2 (bhlhb3 or Sharp 2), and Clock, using β actin as an endogenous control. Maximal intensity of expression was observed in bhlhb3 at 20:00 hr (Zt12); a peak also occurred at the same circadian time, although decreasing in intensity in the following order: Per3, Per1, cry2, clock, and Per2; bhlhb2 and cry1 peaked at 24:00 hr (Zt16). These genes exhibited the minimal level of expression at between 8:00 (Zt0) and 12:00 hrs (Zt4). Our results demonstrate for the first time that the rat mammary gland expresses clock genes that cycle in response to circadian rhythms. This approach will open new venues for understanding the modulation of the response of the mammary gland to hormones under environmental influences such as light, which might act as endocrine modulators (This work was supported by NCI and NIEHS Grant U01 ES012771, Supl.2).
[Proc Amer Assoc Cancer Res, Volume 47, 2006]