GROWTH PLATE RADIATION RESPONSE--MECHANISMS AND THERAPY

骨生长的干扰或停滞长期以来被认为是一种 治疗儿童恶性肿瘤的并发症 放疗 然而. 几乎没有关于 生长板中这种现象的分子机制。 这 信息的缺乏是我们知识上的一个明显空白 关于这个严重的临床问题，并排除发展， 任何改善辐射诱导生长的治疗策略 钢板损伤 在本提案中，我们提供了以下方面的初步数据： 辐射可能破坏特定的 正常细胞因子和生长因子在生长中的作用 板状软骨 研究的长期目标是：（1） 了解生长因子功能的紊乱， 有助于辐射诱导的骨生长停滞，以及（2） 将这些信息转化为临床适用的策略， 可能改善生长的无毒药物 接受恶性肿瘤放疗的儿童的钢板损伤。 使用鸡和大鼠生长板软骨细胞的初步数据 培养和体内模型表明细胞的特定模式， 以及辐照后的分子事件，其中包括 选择性抑制TGF β和PTHrP，主要的有丝分裂原 导致肿瘤细胞增殖和TNF α的不适当表达， 这可能导致过早肥大和凋亡。 的 自分泌促有丝分裂刺激消失， 过早凋亡与观察到的组织学一致。 本研究的具体目标包括：（1）利用我们的 鸡软骨细胞体外模型，初步表征和 确定3种特异性促有丝分裂生长的破坏模式 因子（bFGF、TGF β、PTHrP），以及不适当的激活 一种关键的细胞因子（TNF α）。 (1B)使用 这项对鸟类软骨细胞的初步研究有助于指导我们转向 一种更稳定哺乳动物离体辐射模型的研究 对啮齿类动物软骨细胞生长的影响 它概括了组织的结构和分化 正常大鼠生长板的级联反应。(2A)识别信号 导致生长因子和TNF α紊乱的机制， 通过对2个特定秒的辐射效应的研究， 信使：胞浆钙离子和cAMP/蛋白激酶A，两者都是 它似乎参与了有丝分裂原的抑制和刺激 细胞凋亡。 (2B)在这个辐射损伤的体外模型中，我们 将研究使用视黄酸的反应修饰，以：抑制 细胞溶质钙、TNF α表达和细胞凋亡;以及 刺激bFGF、TGF β和软骨细胞增殖。 (2C)我们 将研究喷替福林作为一种额外反应调节剂， 降低细胞溶质钙和TNF α表达;以及 刺激cAMP和增殖。(3A)相关体外结果 使用基于组织的方法在大鼠体内模型中发现 例如免疫细胞化学和原位杂交， 辐射损伤的机制，以及新的 临床上可应用的治疗策略。

Disturbance or arrest of bone growth has long been recognized as a complication of the treatment of childhood milignancies with radiotherapy. However. There has been almost no work on the molecular mechanism of this phenomenon in the growth plate. This lack of information constitutes a glaring gap in our knowledge about this serious clinical problem, and precludes development of any therapeutic strategies to ameliorate radiation-induced growth plate injury. In the present proposal we present preliminary data on possible mechanisms by which radiation may disrupt specific aspects of normal cytokine and growth factor function in growth plate cartilage. The long term goals of the research are (1) to understand the derangements of growth factor function which contribute to radiation-induced bone growth arrest, and (2) to translate this information to clinically applicable strategies using nontoxic pharmacologic agents to potentially ameliorate gorwth plate injury in children undergoing radiotherapy for malignancies. Preliminary data using chick and rat growth plate chondrocyte culture and in vivo models indicate a specific pattern of the cellular and molecular events following irradiation, among which are the selective suppression of TGFbeta and PTHrP, the major mitogens driving proliferation, and inappropriate expression of TNFalpha, which may lead to premature hypertrophy and apoptosis. The obliteration of the autocrine mitogenic stimulus and evidence of premature apoptosis are consistent with the observed histology. The specific aims of the proposed reserch include: (1A) use our chick chondrocyte in vitro model to initially characterize and determine the pattern of disruptio of 3 specific mitogenic growth factors (bFGF.TFGbeta.PTHrP), and the innappropriate activation of one key cytokine (TNFalpha) following irradiation. (1B) Use this initial study of avian chondrocytes to help guide our shift to study of a more levelvant mammalian in vitro model of radiation effects on devloping rodent chondrocytes, using pellet cultures which recapitulate the tissue arechitecture and differentiation cascade of normal rat growth plate. (2A) Identify the signaling mechanisms leading to growth factor and TNFalpha derangement, through investigation of radiation effects on 2 specific second messengers: cytosolic calcium and cAMP/protein kinase A, both of which appear to be involved in mitogen suppression and stimulation of apoptosis. (2B) In this in vitro model of radiation damage, we will study response modification using retinoic acid to: suppress cytosolic calcium, TNFalpha expression, and apoptosis; and to stimulate bFGF, TFGbeta, and chondrocyte proliferation. (2C) We will study pentoxifylline as an additinal response modifier to: decrease cytosolic calcium and TNFalpha expression; and to stimulate cAMP and proliferation. (3A) Correlate in vitro findings with findings in a rat in vivo model using tissue-based approaches such as immunocytochemistry and in situ hybridization to conform mechanisms of radiation injury to the physis, as well as new clinically aplicable therapeutic strategies.