TY - JOUR
T1 - Rapid detection, cloning and molecular cytogenetic characterisation of sequences from an MRP-encoding amplicon by chromosome microdissection
AU - Ray, M. E.
AU - Guan, X. Y.
AU - Slovak, M. L.
AU - Trent, J. M.
AU - Meltzer, P. S.
N1 - Funding Information:
We gratefully acknowledge Dr S.P.C. Cole and Dr R.G. Deeley for their critical review of the manuscript. We also acknowledge the excelknt technical assistance in chromosome banding by Ann Burgess. Dr M.L. Slovak is a member of the City of Hope Cancer Research Center, which is supported by Public Health Service Grant CIA-33572.
PY - 1994/7
Y1 - 1994/7
N2 - Chromosome microdissection was utilised for the analysis of cytogenetic markers of gene amplification [homogeneously staining regions (hsrs) and double minutes (dmins)] in two doxorubicin-resistant cell lines, fibrosarcoma HT1080/DR4 and small-cell lung cancer H69AR. Microdissection products from the hsr(7)(p12p15) of HT1080/DR4 were amplified and used for fluorescent in situ hybridisation (micro-FISH) analysis of drug-sensitive HT1080, resistant HT1080/DR4 and normal lymphocytes. The results demonstrated that the hsr contains a domain of DNA amplification of complex origin including sequences derived from 16p11.2-16p13.1, 2q11.2, 7q32-7q34 and 10q22. The amplification was confirmed by converting the micro-dissected probe into a microclone library for probing HT1080 and HT1080/DR4 Southerns. A micro-FISH probe from normal band region 16p11-16p13 further demonstrated amplification of 16p sequences in both HT1080/DR4 and H69AR. During the course of this analysis, Cole et al. (1992) (Science, 258, 1650-1653) published the amplification of the MRP gene in H69AR cells, which maps to chromosome 16p13.1. Our results corroborate the finding of MRP amplification in these doxorubicin-resistant cell lines, but, importantly, they provide information on the composition of the complex amplicon contributions from four different chromosomes. This study demonstrates the potential utility of chromosome microdissection for the rapid recovery of sequences from amplified regions in drug-resistant cells.
AB - Chromosome microdissection was utilised for the analysis of cytogenetic markers of gene amplification [homogeneously staining regions (hsrs) and double minutes (dmins)] in two doxorubicin-resistant cell lines, fibrosarcoma HT1080/DR4 and small-cell lung cancer H69AR. Microdissection products from the hsr(7)(p12p15) of HT1080/DR4 were amplified and used for fluorescent in situ hybridisation (micro-FISH) analysis of drug-sensitive HT1080, resistant HT1080/DR4 and normal lymphocytes. The results demonstrated that the hsr contains a domain of DNA amplification of complex origin including sequences derived from 16p11.2-16p13.1, 2q11.2, 7q32-7q34 and 10q22. The amplification was confirmed by converting the micro-dissected probe into a microclone library for probing HT1080 and HT1080/DR4 Southerns. A micro-FISH probe from normal band region 16p11-16p13 further demonstrated amplification of 16p sequences in both HT1080/DR4 and H69AR. During the course of this analysis, Cole et al. (1992) (Science, 258, 1650-1653) published the amplification of the MRP gene in H69AR cells, which maps to chromosome 16p13.1. Our results corroborate the finding of MRP amplification in these doxorubicin-resistant cell lines, but, importantly, they provide information on the composition of the complex amplicon contributions from four different chromosomes. This study demonstrates the potential utility of chromosome microdissection for the rapid recovery of sequences from amplified regions in drug-resistant cells.
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U2 - 10.1038/bjc.1994.254
DO - 10.1038/bjc.1994.254
M3 - Article
C2 - 8018546
AN - SCOPUS:0028180832
SN - 0007-0920
VL - 70
SP - 85
EP - 90
JO - British journal of cancer
JF - British journal of cancer
IS - 1
ER -