Li W, Fan J, Hochhauser D, Banerjee D, Zielinski Z, Almasan A, Yin Y, Kelly R, Wahl G M, Bertino J R
Program for Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10436-40. doi: 10.1073/pnas.92.22.10436.
Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/- cell lines exhibited a 2- to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3- to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.
生长抑制试验表明，在缺乏视网膜母细胞瘤蛋白（pRB）的脂肪肉瘤细胞系HS - 18和具有截短且无功能pRB的骨肉瘤细胞系SaOS - 2中，甲氨蝶呤（MTX）和5 - 氟脱氧尿苷（FdUrd）的IC50值分别比具有野生型pRB的HT - 1080（纤维肉瘤）细胞系高10至12倍和4至11倍。与HT - 1080（Rb +/+）细胞相比，这些Rb -/-细胞系的二氢叶酸还原酶（DHFR）和胸苷酸合成酶（TS）酶活性增加了2至4倍，这些酶的mRNA水平也增加了3至4倍。这种表达增加并非由于DHFR和TS基因的扩增。在SaOS - 2细胞的Rb转染子中，MTX和FdUrd的生长抑制作用增强，DHFR和TS活性及表达相应降低。相比之下，对于非抗代谢物依托泊苷、顺铂和阿霉素，这些细胞系在生长抑制方面没有显著差异。凝胶迁移率变动分析表明，与Rb重组的SaOS - 2细胞相比，亲本SaOS - 2细胞的游离E2F水平升高。这些结果表明，pRB缺陷细胞对由E2F蛋白增强转录的基因所编码的靶酶介导的生长抑制可能敏感性降低，并提示了细胞毒性药物与参与细胞周期进程的基因之间的相互作用机制。
