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Engineering targeted chromosomal amplifications in human breast epithelial cells.

Springer S, Yi KH, Park J, Rajpurohit A, Price AJ, Lauring J - Breast Cancer Res. Treat. (2015)

Bottom Line: We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker.Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH.This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

View Article: PubMed Central - PubMed

Affiliation: The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, CRB 1 Room 146, 1650 Orleans Street, Baltimore, MD, 21287, USA.

ABSTRACT
Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

No MeSH data available.


Related in: MedlinePlus

FISH on parental MCF-7 cells and amplified ZNF703-targeted subclones E8, F3, and G5. Nuclei are stained with DAPI. The green probe is to chromosome 8 centromeric sequences. a The red probe is a BAC in the ZNF703 region on 8p11-12. b The red probe is a BAC in the FGFR1 region
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Fig4: FISH on parental MCF-7 cells and amplified ZNF703-targeted subclones E8, F3, and G5. Nuclei are stained with DAPI. The green probe is to chromosome 8 centromeric sequences. a The red probe is a BAC in the ZNF703 region on 8p11-12. b The red probe is a BAC in the FGFR1 region

Mentions: Because ddPCR and array CGH average copy number over the entire population, we performed FISH to assess copy number changes at the level of individual cells, using probes for centromeric sequences on chromosome 8 and three BAC probes located near FGFR1, at the centromeric end of the 8p11-12 amplification, ZNF703, and NRG1, which is located 5 Mb telomeric to ZNF703. Parental MCF-7, 38C-3, and amplified subclones all showed two signals for centromere 8, and MCF-7 and 38C-3 were diploid for the other loci tested (Fig. 4 and Supplemental Figure 4). Clones E8, F3, and G5 all showed increased FISH signals for ZNF703, consistent with the estimated copy number by ddPCR, and F3 and G5 showed similar increases in signals for FGFR1. Clone E8 showed low level gain of FGFR1, also consistent with the array CGH results (Supplemental Figures 1 and 4). Clone G5 showed only a single copy of NRG1 by FISH, consistent with the telomeric copy number loss observed by array CGH (Supplemental Figures 3 and 4). Clone E8 showed more heterogeneity than clones F3 and G5 at the cellular level, with significant variability of NRG1 copy number among individual cells, possibly indicating a greater degree of genomic instability in this clone (Supplemental Figure 4).Fig. 4


Engineering targeted chromosomal amplifications in human breast epithelial cells.

Springer S, Yi KH, Park J, Rajpurohit A, Price AJ, Lauring J - Breast Cancer Res. Treat. (2015)

FISH on parental MCF-7 cells and amplified ZNF703-targeted subclones E8, F3, and G5. Nuclei are stained with DAPI. The green probe is to chromosome 8 centromeric sequences. a The red probe is a BAC in the ZNF703 region on 8p11-12. b The red probe is a BAC in the FGFR1 region
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491111&req=5

Fig4: FISH on parental MCF-7 cells and amplified ZNF703-targeted subclones E8, F3, and G5. Nuclei are stained with DAPI. The green probe is to chromosome 8 centromeric sequences. a The red probe is a BAC in the ZNF703 region on 8p11-12. b The red probe is a BAC in the FGFR1 region
Mentions: Because ddPCR and array CGH average copy number over the entire population, we performed FISH to assess copy number changes at the level of individual cells, using probes for centromeric sequences on chromosome 8 and three BAC probes located near FGFR1, at the centromeric end of the 8p11-12 amplification, ZNF703, and NRG1, which is located 5 Mb telomeric to ZNF703. Parental MCF-7, 38C-3, and amplified subclones all showed two signals for centromere 8, and MCF-7 and 38C-3 were diploid for the other loci tested (Fig. 4 and Supplemental Figure 4). Clones E8, F3, and G5 all showed increased FISH signals for ZNF703, consistent with the estimated copy number by ddPCR, and F3 and G5 showed similar increases in signals for FGFR1. Clone E8 showed low level gain of FGFR1, also consistent with the array CGH results (Supplemental Figures 1 and 4). Clone G5 showed only a single copy of NRG1 by FISH, consistent with the telomeric copy number loss observed by array CGH (Supplemental Figures 3 and 4). Clone E8 showed more heterogeneity than clones F3 and G5 at the cellular level, with significant variability of NRG1 copy number among individual cells, possibly indicating a greater degree of genomic instability in this clone (Supplemental Figure 4).Fig. 4

Bottom Line: We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker.Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH.This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

View Article: PubMed Central - PubMed

Affiliation: The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, CRB 1 Room 146, 1650 Orleans Street, Baltimore, MD, 21287, USA.

ABSTRACT
Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

No MeSH data available.


Related in: MedlinePlus