MOLECULAR CORRELATES OF METHOTREXATE IN CHILDHOOD ALL

描述：（申请人摘要）申请人以前的研究使用 基于流式细胞术的甲氨蝶呤（MTX）评估方法 少量白血病原始细胞中的耐药/敏感性标志物。 他 获得的证据表明，在新诊断的 涉及二氢叶酸还原酶（DHFR）水平升高的儿童ALL， MTX的靶酶，并在较小程度上降低MTX细胞 还原叶酸载体（RFC）。 最近，他发现， MTX耐药标志物可能是谱系特异性的（DHFR的频率为100%）。 在T细胞中的过度表达远大于B前体ALL）， 可能是由于MTX化疗而获得的（它们的水平 在复发样品中增加）。 在本申请中，申请人提出 (in目的1）建立新的、灵敏度更高的DHFR检测方法 水平和MTX膜转运，包括 免疫荧光测定RT-PCR和（3 H）MTX摄取的直接测定。 他 将T细胞和B前体ALL原始细胞中的这些参数关联起来， 诊断与无事件生存，与白血病复发配对 诊断/复发标本以及与患者和野生病相关的 参数 他将使用这些方法（目标1）来探索 DHFR表达升高或RFC变化之间的关系 水平或结构，以及关键细胞周期控制的分子改变 (p53、p16 INK 4A和MDM 2）参与ALL白血病发生。 后者将 通过包括基因组PRT-PCR的标准分子方法进行测定，和 SSCP-PCR。 p16 INK 4A表达与MT敏感性的关系 和抗性概况，以及抗性机制将进一步研究 在p16 INK 4A阴性T-ALL细胞中，转染p16 INK 4A cDNA表达 结构。 最后，（在目标3中）他将直接考察 B前体ALL患者的良好预后， 21号染色体特异性改变（超二倍体，t（l2;21））部分是由于 RFC表达增加和MTX转运活性升高。 超二倍体患者将通过标准细胞遗传学鉴定， t（12;21）患者将通过TEL/AML 1的RT-PCR测定来鉴定。 转录本; RFC表达和功能将通过 目标1. 将使用表达TEL/AML 1的细胞系进行体外实验。 用于验证患者原始细胞的结果，也将用于研究 RFC表达增加的分子基础。 的结果 拟议的研究将为进一步优化现代ALL提供框架， 包括MTX在内的治疗方法，并促进开发 在遇到阻力时避开它

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.