MOLECULAR CORRELATES OF METHOTREXATE IN CHILDHOOD ALL

描述：(申请者摘要)申请者以前的学习情况 基于流式细胞术的甲氨蝶呤(MTX)检测方法 少量白血病细胞中的耐药/敏感标志物。他 新诊断的甲氨蝶呤固有耐药性的作用获得证据 儿童时期都涉及二氢叶酸还原酶(DHFR)水平升高， MTX的靶酶，并在一定程度上降低了MTX细胞 被还原的叶酸载体(RFC)摄取。最近，他发现 MTX抗性标记可能是谱系特异性的(DHFR的常见 T细胞的过度表达远远大于B-前体ALL)，而且 可能是由于使用MTX化疗而获得的(他们的水平 复发样本增加)。在本申请书中，申请人建议 (目标1)开发新的、更灵敏的DHFR检测方法 急性淋巴细胞白血病儿童的MTX水平和膜转运，包括 免疫荧光法、RT-PCR法和(~3H)MTX摄取直接法。他 会将T细胞和B细胞前体细胞的这些参数关联起来 诊断为无事件生存，配对的白血病复发 诊断/复发标本以及与患者和野生疾病相关的 参数。他将使用这些方法(在目标1中)探索 DHFR表达升高与RFC变化的关系 关键细胞周期调控中的水平或结构以及分子变化 (p53、p16INK4A和MDM2)与所有白血病的发生有关。后者将 通过包括基因组PRT-PCR在内的标准分子方法进行检测，以及 SSCP-聚合酶链式反应。P16INK4A基因表达与MT敏感性的关系 和抗性分布，并进一步研究抗性机制。 P16INK4A基因在p16INK4A阴性T-ALL细胞中的表达 构造。最后，(在目标3)他将直接研究这种可能性 B-前体患者的良好预后都表现为原始细胞 21号染色体特有的改变(超二倍体，t(L2；21))是部分原因 与RFC表达增加和MTX转运活性升高有关。 超二倍体患者将通过标准细胞遗传学进行鉴定，而 T(L2；21)患者将通过TEL/AML1的RT-PCR检测来识别 转录本；RFC的表达和功能将通过 目的1.用TEL/AML1表达的细胞株进行体外实验 用于验证患者爆炸的结果，也将用于研究 RFC表达增加的分子基础。评选结果 拟议的研究将为进一步优化现代ALL提供一个框架， 包括甲氨蝶呤在内的疗法，并促进开发治疗 当阻力确实出现时，绕过它。

DESCRIPTION: (Applicant's Abstract) The applicant's previous studies used flow cytometry based methodologies for assessing methotrexate (MTX) resistance/sensitivity markers in small numbers of leukemic blasts. He obtained evidence for a role for inherent MTX resistance in newly diagnosed childhood ALL involving increased levels of dihydrofolate reductase(DHFR), the target enzyme for MTX, and to a minor extent decreased MTX cellular uptake by the reduced folate carrier (RFC). More recently, he found that MTX resistance markers could be lineage specific (the frequent of DHFR overexpression was far greater in T-cell than B-precursor ALL) and also could be acquired as a result of chemotherapy with MTX (their levels increased in relapsed samples). In this application the applicant proposes (in Aim 1) to develop new and more sensitive methods for assaying DHFR levels and MTX membrane transport in children with ALL, including immunofluorescence assay RT-PCR, and direct assays of (3H)MTX uptake. He will correlate these parameters in T-cell and B-precursor ALL blasts at diagnosis with event-free survival, with leukemic relapse in paired diagnosis/relapse specimens and with patient and wild disease related parameters. He will use these methods (in Aim 1) to explore the relationships between the expression of elevated DHFR, or changes in RFC levels or structure, and molecular alterations in key cell cycle controls (p53, p16INK4A, and MDM2) implicated in ALL leukemogenesis. The latter will be assayed by standard molecular approaches including genomic PRT-PCR, and SSCP-PCR. The relationship between p16INK4A expression and MT sensitivity and resistance profiles, and resistance mechanisms will be further studied in p16INK4A negative T-ALL cells transfected with p16INK4A cDNA expression constructs. Finally, (in Aim 3) he will directly examine the possibility that good prognoses of patients with B-precursor ALL whose blast exhibit chromosome 21-specific alterations (hyperdiploidy, t(l2;21)) are partly due to increased expression of RFC and elevated MTX transport activity. Hyperdiploid patients will be identified by standard cytogenetics whereas t(l2;21) patients will be identified by RT-PCR assay of the TEL/AML1 transcript; RFC expression and function will be measured by the methods in Aim 1. In vitro experiments with TEL/AML1-expressing cell lines will be used to verify the results in patient blasts and will also be used to study the molecular basis for increased RFC expression. The results of the proposed studies will provide a framework for further optimizing modern ALL, therapies including MTX and facilitate the development of approaches for circumventing resistance when it does arise.