Ray M E, Su Y A, Meltzer P S, Trent J M
Department of Human Genetics, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Oncogene. 1996 Jun 20;12(12):2527-33.
Melanocytic transformation is thought to occur by the sequential accumulation of genetic alterations. Evidence implicating human chromosomes as a site for a gene(s) involved in melanoma suppression comes from studies of LOH [loss of heterozygosity], cytogenetics and biologic reversion of tumorigenicity following the introduction of a normal chromosome 6 by microcell-mediated chromosome transfer (Trent et al., 1990). Using a tumorigenic melanoma cell line (UACC 903) and a chromosome-6 suppressed melanoma subline [UACC 903 (+6)], we have isolated a series of genes uniquely expressed in the suppressed subline. A modified PCR-based cDNA subtraction technique was used to generate subtracted cDNA sublibraries for both the parental and (+6) suppressed cells. A total of 32 randomly selected clones from the suppressed sublibrary were isolated and examined, with 24 detecting a transcript by Northern analysis. Of these 24 clones, 21 (88%) demonstrated elevated expressed by Northern analysis in the suppressed subline relative to the tumorigenic parental cell line. In 6/21 differentially expressed clones (29%), expression was exclusive to the suppressed subline. Partial sequence analysis and database searching of these clones indicated that 5/6 were novel with one representing a previously characterized gene. Chromosomal localization of the five novel clones was performed following PCR amplification of a human/rodent somatic cell hybrid mapping panel or fluorescent in situ hybridization. One cDNA (termed AIM1) was localized to a band-region of chromosome 6 frequently deleted in melanomas (6q21). This novel approach should facilitate the identification of genes whose expression is causally related to the suppressed phenotype.
黑素细胞转化被认为是通过基因改变的逐步积累而发生的。涉及人类染色体作为参与黑色素瘤抑制的一个或多个基因位点的证据来自杂合性缺失（LOH）研究、细胞遗传学以及通过微细胞介导的染色体转移引入正常6号染色体后肿瘤发生性的生物学逆转研究（特伦特等人，1990年）。使用致瘤性黑色素瘤细胞系（UACC 903）和6号染色体抑制的黑色素瘤亚系[UACC 903（+6）]，我们分离出了一系列在抑制亚系中独特表达的基因。采用一种基于PCR的改良cDNA消减技术，为亲代细胞和（+6）抑制细胞生成消减cDNA亚文库。从抑制亚文库中总共随机分离并检测了32个克隆，其中24个通过Northern分析检测到转录本。在这24个克隆中，21个（88%）通过Northern分析显示在抑制亚系中相对于致瘤性亲代细胞系表达升高。在21个差异表达克隆中的6个（29%）中，表达仅见于抑制亚系。对这些克隆进行部分序列分析和数据库搜索表明，6个中有5个是新的，其中一个代表一个先前已鉴定特征的基因。在对人/啮齿动物体细胞杂交定位板进行PCR扩增或荧光原位杂交后，对这5个新克隆进行了染色体定位。一个cDNA（称为AIM1）定位于黑色素瘤中经常缺失的6号染色体的一个条带区域（6q21）。这种新方法应有助于鉴定其表达与抑制表型有因果关系的基因。
