Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization.
Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-alpha (TNF-alpha). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na+/K+-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.
Boukerche
Habib
H
Department of Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, NY 10032, USA.
Su
Zao-Zhong
ZZ
Kang
Dong-Chul
DC
Fisher
Paul B
PB
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D015723:Gene Library; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D009362:Neoplasm Metastasis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014183:Transplantation, Heterologous
D000254:Sodium-Potassium-Exchanging ATPase