Cignetti A, Guarini A, Carbone A, Forni M, Cronin K, Forni G, Gansbacher B, Foa R
Dipartimento di Scienze Biomediche ed Oncologia Umana, Sezione Clinica, University of Turin, Italy.
J Natl Cancer Inst. 1994 May 18;86(10):785-91. doi: 10.1093/jnci/86.10.785.
Previous studies have suggested that some of the limitations associated with the administration of high-dose exogenous interleukin 2 (IL2) may be overcome, at least partly, by cytokine gene transfer modalities. These findings have prompted investigations into whether human tumor cells may be transduced with the IL2 gene and whether tumor cell lines could be engineered to release IL2.
The purpose of this study was to evaluate the possibility of inducing a productive transfer of the IL2 gene into human acute leukemia cells and to assess the phenotypic and proliferative changes generated in the engineered cells, as well as their tumorigenic potential in nude mice.
Three retroviral vectors (DC/TK/IL2, DC/AD/R/IL2, and N2/CMV/IL2) carrying the IL2 gene were used to transduce three human leukemic cell lines: K562 and U937 (myeloid) and ST4 (lymphoid). Messenger RNA expression of the IL2 gene was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and productive IL2 release using a human IL2 assay and an enzyme-linked immunosorbent assay kit. The expression of the p55 (alpha) and p75 (beta) chains of the IL2 receptor were determined by RT-PCR and indirect immunofluorescence. The kinetics of in vitro growth and proliferation of parental and engineered cells were also measured. Parental and IL2 gene-transduced ST4 lymphoblasts were injected into immunosuppressed nude mice that had their tumors measured twice weekly.
The productive insertion of the IL2 gene was achieved in all three cell lines studied. The amounts of IL2 constitutively released by the engineered neoplastic cells ranged between 1 and 11 U/mL of IL2 produced from 10(6) cells in 72 hours. A fivefold increase in IL2 production was obtained in ST4 cells by further limiting dilution cloning of the bulk-infected cells. The stable integration of the IL2 gene did not modify the phenotype of the leukemic cells, the expression of the IL2 receptor alpha and beta chains and of several cytokine genes, or the kinetics of in vitro growth and proliferation. In nude mice injected with various IL2-producing ST4 clones, tumor growth associated inversely with the amounts of IL2 secreted by the leukemic cells.
The results of this study demonstrate that the IL2 gene can be productively transduced into human myeloid and lymphoid leukemic cells without modifying their phenotypic and proliferative properties and that this transduction leads to a reduced or abrogated in vivo tumorigenic potential.
先前的研究表明，与高剂量外源性白细胞介素2（IL2）给药相关的一些局限性，至少可以部分地通过细胞因子基因转移方式来克服。这些发现促使人们研究人肿瘤细胞是否可以用IL2基因进行转导，以及肿瘤细胞系是否可以被改造以释放IL2。
本研究的目的是评估将IL2基因有效转移到人类急性白血病细胞中的可能性，并评估工程细胞中产生的表型和增殖变化，以及它们在裸鼠中的致瘤潜力。
使用三种携带IL2基因的逆转录病毒载体（DC/TK/IL2、DC/AD/R/IL2和N2/CMV/IL2）转导三种人类白血病细胞系：K562和U937（髓系）以及ST4（淋巴系）。通过逆转录聚合酶链反应（RT-PCR）分析IL2基因的信使RNA表达，并使用人IL2检测法和酶联免疫吸附测定试剂盒检测IL2的有效释放。通过RT-PCR和间接免疫荧光法测定IL2受体的p55（α）和p75（β）链的表达。还测量了亲本细胞和工程细胞的体外生长和增殖动力学。将亲本和IL2基因转导的ST4淋巴母细胞注射到免疫抑制的裸鼠中，每周两次测量其肿瘤大小。
在所研究的所有三种细胞系中均实现了IL2基因的有效插入。工程化肿瘤细胞组成性释放的IL2量在每72小时从10^6个细胞产生的1至11 U/mL IL2之间。通过对大量感染细胞进行进一步的有限稀释克隆，ST4细胞中的IL2产量增加了五倍。IL2基因的稳定整合并未改变白血病细胞的表型、IL2受体α和β链以及几种细胞因子基因的表达，也未改变体外生长和增殖动力学。在注射了各种产生IL2的ST克隆的裸鼠中，肿瘤生长与白血病细胞分泌的IL2量呈负相关。
本研究结果表明，IL2基因可以有效地转导到人类髓系和淋巴系白血病细胞中，而不会改变它们的表型和增殖特性，并且这种转导会导致体内致瘤潜力降低或消除。
