Transforming DNA sequences present in human prolactin-secreting pituitary tumors.
Five human PRL-secreting pituitary tumors were tested for the presence of DNA-transforming sequences. After calcium phosphate transfection to NIH-3T3 mouse fibroblast cells, DNA samples derived from two prolactinomas induced foci of morphologically transformed cells which subsequently grew in soft agar. After retransfection of transformant DNA, resulting secondary transformants elicited rapidly growing solid tumors in nude mice. Southern analysis of transformant DNA revealed the integration of Alu-positive human DNA sequences into the mouse fibroblast NIH-3T3 cells, as judged by hybridization to a Blur-8 probe. The Alu signal became increasingly more difficult to detect with the multiple passaging (greater than 20) of transformant cells in culture. Alu polymerase chain reaction (PCR) was, therefore, used to selectively amplify human DNA sequences from the NIH-3T3 rodent background. PCR using a human Alu-specific primer resulted in amplification of an Alu-containing DNA region within these transformants. The transformant DNA did not hybridize to human genomic probes for genes known to evoke focus formation in this assay, including H-ras, K-ras, N-ras, trk, ret, ros, or met. Further identification of the Alu-containing region revealed that it contained sequences from the human hst gene, a member of the fibroblast growth factor family. The presence of human hst was demonstrated by strong hybridization to a 40-mer oligonucleotide probe to the second exon of hst, by amplification of this region with human hst-nested amplimers within the first and second introns, and finally by direct sequencing.(ABSTRACT TRUNCATED AT 250 WORDS)
Gonsky
R
R
Department of Medicine, Cedars-Sinai Medical Center-University of California School of Medicine, Los Angeles 90048.
Herman
V
V
Melmed
S
S
Fagin
J
J
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002471:Cell Transformation, Neoplastic; D004273:DNA, Neoplasm / Q000235:genetics; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors; D006801:Humans; D007438:Introns; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D009838:Oligodeoxyribonucleotides; D009857:Oncogenes; D010911:Pituitary Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015175:Prolactinoma / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004273:DNA, Neoplasm; C496445:FGF4 protein, human; C496446:Fgf4 protein, mouse; D051519:Fibroblast Growth Factor 4; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005346:Fibroblast Growth Factors