Cell cultureMethylene blue Cell growth Cytotoxicity A B S T R A C T Cell counting is a common technique in cellular and molecular biology research applications, such as cell culture maintenance, cell plating, cell growth and cell doubling time determinations, as well as cell proliferation and cytotoxicity measurements. Many commonly employed cell counting methods exhibit limitations that influence resulting accuracy or versatility. For example, the trypan blue method typically underestimates cell numbers in culture, and the Lowry protein assay can be influenced by cell cycle. An urgent need exists for a method of cell counting that is both accurate and versatile. This work intended to explore an adaptation of the methylene blue assay to overcome the existing limitations of the procedure, enabling application to a broader range of cell densities and various cell culture plates. This new methylene blue assay was found to be more efficient, accurate and sensitive. A linear relationship (r 2 > 0.99) was established between cell number and absorbance at 570 nm wavelength when the new methylene blue assay was applied to three cell lines (HepG2, Caco-2, and MCF-7) plated in a broad range of cell densities (5 · 10 4 to 2.5 · 10 6 ) in four different types of culture plates (6-, 12-, 24-, and 96-well plates).Growth curves were determined using both the trypan blue and methylene blue methods.At each time point in the HepG2 growth curve, the cell count obtained using the trypan blue assay was statistically significantly lower than that obtained using the methylene blue assay (p < 0.05). The same was true for the Caco-2 growth curve at all time points (p < 0.05) except at the 0 h. The methylene blue method proposed in this paper may serve as a direct, automated counting method for cells grown in any type of culture plates. This assay has clear advantages over traditional methods and is a powerful tool for any application requiring a versatile, efficient, and accurate method of cell counting, such as bioavailability and cytotoxicity assays, and more basic experiments such as cell growth curve or doubling time determination, especially in the research of natural products, bioactive compounds, phytochemicals, functional foods and nutraceuticals.

Estrogen action in mammary gland development and breast cancer progression is tightly linked to the GH/IGF-I axis. Although many of the effects of GH on mammary gland growth and development require IGF-I, the extent to which GH action in breast cancer depends on IGF-I is not known. We examined GH action in a panel of estrogen receptor-positive breast cancer cell lines and found that T47D cells express significant levels of GH receptor and that GH significantly enhances 17β-estradiol (E2)-stimulated proliferation in these cells. GH action in the T47D cells was independent of changes in IGF-I and IGF-I receptor (IGF-IR) expression and IGF-IR signaling, suggesting that GH can exert direct effects on breast cancer cells. Although E2-dependent proliferation required IGF-IR signaling, the combination of GH+E2 overcame inhibition of IGF-IR activity to restore proliferation. In contrast, GH required both Janus kinase 2 and epidermal growth factor receptor signaling for subsequent ERK activation and potentiation of E2-dependent proliferation. Downstream of these pathways, we identified a number of immediate early-response genes associated with proliferation that are rapidly and robustly up-regulated by GH. These findings demonstrate that GH can have important effects in breast cancer cells that are distinct from IGF-IR activity, suggesting that novel drugs or improved combination therapies targeting estrogen receptor and the GH/IGF axis may be beneficial for breast cancer patients.

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Estrogen receptor (ER) and insulin-like growth factor-I receptor (IGF-IR) interact and activate one another in breast cancer cells, and ER can up-regulate components of the IGF-I signaling pathway. Therapeutic strategies co-targeting ER and IGF-IR in ER-positive breast tumors have been developed, yet clinical trials of anti-IGF-IR therapies have not been as successful as had been hoped. Although growth hormone (GH) is implicated in having a role in breast cancer, its effects are often attributed to the actions of IGF-I. Since inhibition of IGF-IR can cause an increase in circulating levels of GH, we considered the possibility that GH may act directly on the tumor itself thus explaining the limited efficacy of anti-IGF-IR therapy in breast cancer. Therefore, we sought to determine whether GH has direct, IGF-I-independent effects on breast cancer. We demonstrate in T47D human breast cancer cells that while GH alone only weakly stimulates cell proliferation, it significantly enhances estradiol (E2)-stimulated proliferation. Inhibition of IGF-IR reduced E2-stimulated proliferation, confirming previous findings. Remarkably, GH+E2 overcame the IGF-IR blockade to promote proliferation. This indicates that GH not only acts in an IGF-I-independent manner, but can bypass IGF-IR inhibitors to restore E2 action on proliferation. Furthermore, we found that both epidermal growth factor receptor (EGFR) and ERK were required for proliferation stimulated by GH and E2, and that GH uses EGFR to activate ERK even in the presence of the IGF-IR inhibitor. Expression of proliferation genes up-regulated by GH+E2 treatment was abrogated by the EGFR and MEK inhibitors, but not by the IGF-IR inhibitor. Thus, we propose that while E2-stimulated proliferation requires IGF-IR, EGFR, and ERK, the presence of GH allows E2 to bypass the requirement for IGF-IR while remaining dependent on EGFR and ERK. Furthermore, we have found that GH can potentiate E2 stimulation of ER target gene transcription and this is dependent on ERK, but not on IGF-IR. Taken together, these findings indicate that GH not only can act directly on breast cancer cells independently of IGF-I, but can bypass IGF-IR blockade to stimulate ERK and potentiate E2 activity on proliferation and gene regulation. A better understanding of the interactions between GH and E2 will contribute to the development of novel drugs or improved combination therapies targeting the GH/IGF/E2 axis in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3923. doi:1538-7445.AM2012-3923

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