Prévostel C, Alvaro V, de Boisvilliers F, Martin A, Jaffiol C, Joubert D
INSERM U401, Montpellier, France.
Oncogene. 1995 Aug 17;11(4):669-74.
An altered protein expression of Ca(2+)-dependent protein kinase C (PKC) isoforms and a point mutation in the PKC alpha cDNA (position 908 of the nucleotide sequence, position 294 of the amino acid sequence, substitution of an aspartic acid by a glycine) have been previously described in a subpopulation of human pituitary tumors. In this work, we screened 16 thyroid tissue samples (four follicular adenomas, five colloid adenomas, three papillary carcinomas, one follicular carcinoma and three normal tissues adjacent to the tumors) for the presence of the PKC alpha point mutation and for PKC alpha, beta 1, beta 2, epsilon and delta protein expression. Screening for the presence of the PKC alpha mutant was performed by a subcloning technic. The polymerase chain reaction products were generated using reverse-transcribed cDNAs, subcloned and sequenced (10 clones were routinely sequenced). The PKC alpha point mutation at position 908 of the cDNA sequence was found in four out of the nine adenomas and in the follicular carcinoma. It was neither detected in the papillary carcinomas nor in the adjacent normal tissues (one was the adjacent normal tissue of the follicular carcinoma; in this sample, genomic DNA and cDNA were used to look for the presence of the mutant), demonstrating the somatic nature of this mutant. Western blot analysis of PKC isoforms showed that the expression of all isoforms was higher in the thyroid neoplasms as compared with their adjacent normal tissue (n = 3). It was also higher in the samples containing the PKC mutant (two follicular adenomas, two colloid adenomas and the follicular carcinoma) as compared with the tumors where it was not detected (three papillary carcinomas and five adenomas). Samples could be ordered according to their increasing PKC expression as follows: normal adjacent tissue < follicular adenomas without PKC alpha mutant < or = papillary carcinoma < follicular adenomas with PKC mutant < follicular carcinoma with PKC mutant. In conclusion, the discovery of the PKC alpha mutant in thyroid neoplasms demonstrates that this mutant is not particular to human pituitary tumors where it was originally detected. It is a somatic mutation and its presence is concomitant with high levels of all of the PKC isoforms analysed. The presence of the PKC mutant in thyroid neoplasms raises the question of its importance in thyroid tumorigenesis.
先前在一部分人类垂体肿瘤中已描述了钙依赖性蛋白激酶C（PKC）亚型的蛋白表达改变以及PKCα cDNA中的一个点突变（核苷酸序列第908位，氨基酸序列第294位，天冬氨酸被甘氨酸取代）。在本研究中，我们筛查了16份甲状腺组织样本（4份滤泡性腺瘤、5份胶样腺瘤、3份乳头状癌、1份滤泡状癌以及3份肿瘤旁正常组织），以检测PKCα点突变的存在情况以及PKCα、β1、β2、ε和δ蛋白的表达。通过亚克隆技术筛查PKCα突变体的存在情况。使用逆转录cDNA生成聚合酶链反应产物，进行亚克隆并测序（常规对10个克隆进行测序）。在9份腺瘤中的4份以及滤泡状癌中发现了cDNA序列第908位的PKCα点突变。在乳头状癌以及相邻正常组织中均未检测到该突变（其中1份是滤泡状癌的相邻正常组织；在该样本中，使用基因组DNA和cDNA来寻找突变体的存在），这证明了该突变体的体细胞性质。PKC亚型的蛋白质印迹分析表明，与相邻正常组织（n = 3）相比，所有亚型在甲状腺肿瘤中的表达均更高。与未检测到该突变的肿瘤（3份乳头状癌和5份腺瘤）相比，在含有PKC突变体的样本（2份滤泡性腺瘤、2份胶样腺瘤和滤泡状癌）中其表达也更高。样本可根据PKC表达升高情况排序如下：肿瘤旁正常组织＜无PKCα突变体的滤泡性腺瘤＜或 = 乳头状癌＜有PKC突变体的滤泡性腺瘤＜有PKC突变体的滤泡状癌。总之，在甲状腺肿瘤中发现PKCα突变体表明该突变体并非最初检测到它的人类垂体肿瘤所特有。它是一种体细胞突变，其存在与所分析的所有PKC亚型的高水平表达相伴。甲状腺肿瘤中PKC突变体的存在引发了其在甲状腺肿瘤发生中重要性的问题。
