Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation.
A rapid, simple and non-toxic procedure for the simultaneous isolation of DNA and RNA from tumor tissue and cells grown in vitro is described. Guanidinium isothiocyanate was used for homogenization of tumor tissue and for cell lysis. Separation of proteins, DNA and RNA was carried out by isopycnic centrifugation in cesium trifluoroacetate. DNA was further purified by salting out residual protein. Nucleic acids prepared by this method from 47 primary human carcinomas and 17 human cell lines were analysed for amplification and expression of the HER-2/neu proto-oncogene. 2- to 10-fold amplification of HER-2/neu was noted in 7/22 mammary carcinomas (32%) and in 4/14 ovarian carcinomas (28%). No amplification of the proto-oncogene was found in 4 laryngeal carcinomas, 1 pharyngeal carcinoma, 2 retrolingual carcinomas, 3 gastric carcinomas and 1 kidney carcinoma. HER-2/neu overexpression was observed in 6/22 of mammary carcinomas (27%) and 7/14 of ovarian carcinomas (50%). No overexpression was found in all other carcinomas studied. Concordance between amplification and overexpression was noted in 3 mammary and 4 ovarian carcinomas, respectively. 3 mammary and 3 ovarian carcinomas showed overexpression without amplification. 5 human mammary carcinoma cell lines showed both amplification and overexpression of HER-2/neu. In two mammary carcinoma cell lines (MDA MB-453 and ZR 75-1) overexpression was noted without amplification of the proto-oncogene. These data combine to suggest that mechanisms other than gene amplification may also lead to overexpression of the HER-2/neu protooncogene in cancer cells.
Ludwig Boltzmann Institute for Prenatal and Experimental Genome Analysis, Vienna, Australia.
F D
FD
Kury
C
C
Schneeberger
G
G
Sliutz
E
E
Kubista
H
H
Salzer
M
M
Medl
S
S
Leodolter
H
H
Swoboda
R
R
Zeillinger
J
J
Spona
D015139:Blotting, Southern; D001943:Breast Neoplasms / Q000235:genetics; D002460:Cell Line; D002500:Centrifugation, Isopycnic; D004247:DNA / Q000302:isolation & purification; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D010051:Ovarian Neoplasms / Q000235:genetics; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D018719:Erb-b2 Receptor Tyrosine Kinases
D004247:DNA; D004273:DNA, Neoplasm; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D012313:RNA; D012334:RNA, Neoplasm; D018719:Erb-b2 Receptor Tyrosine Kinases; C000715470:MAS1 protein, human