A role of balanced sex hormone in DNA repair in human melanocytes

黑色素瘤发病率的持续上升表明，新的预防策略可能是 除了现有的避免紫外线照射的方法外，还需要其他方法。我们的长期目标是发展这种预防 基于对正常人性别分化DNA损伤修复的全面了解的策略 人类黑素细胞。这项R21提案的目标是确定UVR的性激素调节- 体内外诱导人黑素细胞DNA损伤修复。我们的初步流行病学数据 显示唾液睾酮(T)水平和T/E_2(雌二醇)水平与患黑色素瘤的风险呈负相关； 而我们的细胞生物学数据显示，在培养液中添加睾丸素可以促进正常 人类黑素细胞。我们的中心假设是，较高的睾丸素水平，或较高的T/E2比率，可以保护 涉及PARP1的紫外线诱导DNA损伤的NHMS。我们还假设来自男性和 女性需要不同的T/E_2平衡，因为T/E_2水平在两性之间明显不同。二 提出了具体目标：具体目标1：确定T和T/E2在DNA损伤反应中的作用 在男性和女性的NHMS(活体研究)中。将招募人类受试者，并对皮肤进行治疗 在没有和存在局部应用睾酮的情况下模拟太阳紫外线辐射(改变T或 T/E_2比值)。将监测DNA损伤标记物、激素受体(AR和GPER1)和PARP1水平 在一段时间后，将比较不同条件下标记阳性细胞的比例 黑素细胞和其他皮肤细胞。角质形成细胞和黑素细胞的相互作用将受性激素的影响 由肿瘤坏死因子α和肿瘤坏死因子受体1检测。特异性目标2：检测T/E_2对培养的黑素细胞的影响 男性或女性来源，并在体外确定性激素调节的DNA修复途径。荷尔蒙 在UVR治疗前，将按不同的比例添加激素，以检测效果。基因敲除 AR和GPER1，或AR特异性抑制剂或激动剂将被用来操纵T/AR和E2/GPER1，以及 将研究DNA损伤反应和信号通路。创新之处：据我们所知， 这项研究首次假设T和E2的平衡对使用T/AR预防皮肤黑色素瘤至关重要 系统通过增强DNA修复能力而发挥肿瘤抑制作用。这项拟议的研究具有重要意义 因为它有望通过增加新的参数来改变黑色素瘤预防的范式，例如 性激素测试用于风险评估，或通过饮食或补充剂调整激素水平以进行预防。如果 它的成功，有望导致预防从目前的避免UVR向未来的范式转变 “避免紫外线照射加激素平衡”。

The continuous increase in incidence rates of melanoma suggests novel prevention strategies may be needed in addition to current UVR-avoidance methods. Our long-term goal is to develop such prevention strategies based on a comprehensive understanding of the sex differentiated DNA damage repair in normal human melanocytes. The objective of this R21 proposal is to determine the sex hormonal regulation of UVR- induced DNA damage repair in human melanocytes in vitro and in vivo. Our preliminary epidemiology data showed that salivary testosterone (T) level and T/E2 (estradiol) levels is inversely correlated with melanoma risk; while our cell biology data showed that addition of testosterone in culture media enhanced DNA repair in normal human melanocytes. Our central hypothesis is that higher testosterone levels, or higher T/E2 ratios, protect NHMs from UV-induced DNA damage involving PARP1. We also hypothesize that NHMs from males and females require a different balanced T/E2, as the T/E2 levels apparently dramatically differ in the two sexes. Two specific aims are proposed: Specific Aim 1: To determine the T and T/E2 effects in DNA damage responses in NHMs in men and women (in vivo study). Human subjects will be recruited, and skin will be treated with solar simulated UV radiation in the absence and presence of topical application of testosterone (to alter T or T/E2 ratios). DNA damage markers, hormone receptors (AR and GPER1) and PARP1 levels will be monitored following a time course and percentage of marker positive cells will be compared in different conditions in melanocytes and other skin cells. The keratinocytes-melanocytes interaction impacted by sex hormones will be examined by TNFα and TNFR1. Specific Aim 2: To test T/E2 effect in cultured melanocytes derived from male or female origins and to determine sex hormone-modulated DNA repair pathway in vitro. Hormonal effect will be detected by inclusion of the hormones at various ratios before UVR treatment. Gene knockdown of AR and GPER1, or AR-specific inhibitors or agonists will be used to manipulate T/AR and E2/GPER1, and DNA damage responses and signal pathways will be examined. The innovation: to the best of our knowledge, this study is the first to hypothesize a balanced T and E2 is critical for skin melanoma prevention with T/AR system acting as a tumor suppressor through enhancing DNA repair capacity. The proposed study is significant because it is expected to make a paradigm change in melanoma prevention by adding new parameters such as sex hormone test for risk assessment, or adjusting hormone levels through diet or supplement for prevention. If successful, it is expected to lead to a paradigm change of the prevention from current “avoiding UVR” to future “avoiding UVR plus hormone balance”.