Quinn K A, Treston A M, Unsworth E J, Miller M J, Vos M, Grimley C, Battey J, Mulshine J L, Cuttitta F
Biomarkers and Prevention Research Branch, NCI, National Institutes of Health, Rockville, Maryland 20850, USA.
J Biol Chem. 1996 May 10;271(19):11477-83. doi: 10.1074/jbc.271.19.11477.
The insulin-like growth factors (IGFs), IGF-I and IGF-II, are potent mitogens for human lung and other epithelial cancer cell lines. Previous studies in defined medium lacking added IGF or insulin suggest that an IGF-related ligand can act as an autocrine growth factor for many cancer cell lines through action via the type I IGF receptor (IGF-R). Analysis of RNA isolated from human lung and breast cancer cell lines by reverse transcription of mRNA and polymerase chain reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12 small cell lung cancer cell lines (SCLC), 13/14 nonsmall cell lung cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast lines. All cell lines expressed IGF-R. For analysis of IGF peptide secretion, cell lines were adapted to growth in serum/hormone-free culture medium (R0), and to avoid interference by IGF-binding proteins, secreted IGF peptides were isolated under acidic conditions and analyzed by Western blotting. Based upon measurement of the sensitivity of the anti-IGF antibodies for detection of recombinant human IGFs, IGF peptides accumulated in conditioned medium at greater than picomolar concentrations should have been readily detected. In three cell lines (two lung and one breast) secreted IGF immunoreactivity was detected as three molecular mass species of 23, 14, and 6 kDa. Isolation and NH2-terminal sequencing of each of these species definitively identified them as differentially processed forms of the IGF-II prohormone. Despite the high frequency of IGF-I gene expression detected by reverse transcription-polymerase chain reaction analysis, only one lung cancer cell line, NCI-N417d, was found that unequivocally secreted IGF-I peptide. This direct sequence determination unambiguously identifies IGF-II as the predominant IGF involved in the autocrine growth stimulation of human lung and breast epithelial tumor cell lines and supports a growing body of literature that implicates IGF-II/IGF-R autocrine loops as a common growth mechanism in epithelial carcinogenesis.
胰岛素样生长因子（IGFs），即IGF-I和IGF-II，是人类肺癌及其他上皮癌细胞系的强效促有丝分裂原。先前在缺乏添加IGF或胰岛素的限定培养基中的研究表明，一种与IGF相关的配体可通过I型IGF受体（IGF-R）的作用，作为许多癌细胞系的自分泌生长因子。通过对mRNA进行逆转录和聚合酶链反应，对从人肺癌和乳腺癌细胞系中分离的RNA进行分析，结果显示在大多数细胞系中，IGF-I和IGF-II mRNA与IGF-R共同表达。在12个小细胞肺癌细胞系（SCLC）中的11个、14个非小细胞肺癌（NSCLC）细胞系中的13个以及2个乳腺癌细胞系中的1个中检测到了IGF-I mRNA。在10个SCLC中的8个、12个NSCLC细胞系中的11个以及2个乳腺癌细胞系中的2个中检测到了IGF-II mRNA。所有细胞系均表达IGF-R。为了分析IGF肽的分泌情况，使细胞系适应在无血清/无激素的培养基（R0）中生长，并且为了避免IGF结合蛋白的干扰，在酸性条件下分离分泌的IGF肽，并通过蛋白质印迹法进行分析。基于对抗IGF抗体检测重组人IGF的灵敏度的测定，条件培养基中积累的浓度高于皮摩尔的IGF肽应该很容易被检测到。在三个细胞系（两个肺癌细胞系和一个乳腺癌细胞系）中，检测到分泌的IGF免疫反应性呈现为分子量分别为23、14和6 kDa的三种分子形式。对这些分子形式分别进行分离和氨基末端测序，明确将它们鉴定为IGF-II前激素的不同加工形式。尽管通过逆转录-聚合酶链反应分析检测到IGF-I基因表达的频率很高，但仅发现一个肺癌细胞系NCI-N417d明确分泌IGF-I肽。这种直接的序列测定明确将IGF-II鉴定为参与人肺癌和乳腺上皮肿瘤细胞系自分泌生长刺激的主要IGF，并支持了越来越多的文献观点，即IGF-II/IGF-R自分泌环是上皮癌发生中的一种常见生长机制。
