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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

Copy number profile of chromosome 8 by array CGH. From top to bottom, MCF-7, the targeted, non-amplified MCF-7 clone 38C-3, and the mizoribine-amplified clones E8, F3, and G5. The y-axis represents log2 ratios of copy number, with 0 representing diploid copy number. Red boxes copy number gain, Green boxes copy number loss. The ZNF703 locus is indicated by an arrow. Copy number profiles did not differ from parental MCF-7 cells for the remaining chromosomes (not shown)
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Fig3: Copy number profile of chromosome 8 by array CGH. From top to bottom, MCF-7, the targeted, non-amplified MCF-7 clone 38C-3, and the mizoribine-amplified clones E8, F3, and G5. The y-axis represents log2 ratios of copy number, with 0 representing diploid copy number. Red boxes copy number gain, Green boxes copy number loss. The ZNF703 locus is indicated by an arrow. Copy number profiles did not differ from parental MCF-7 cells for the remaining chromosomes (not shown)

Mentions: To determine the extent and pattern of amplification, we performed genome-wide array CGH on the pre-amplified cells and amplified subclones (Fig. 3 and Supplemental Figures 1–3). As expected from the ddPCR result, all three subclones showed increased copy number of the ZNF703 locus. However, all three clones showed unique patterns of copy number change at surrounding loci. Clones F3 and G5 showed broad, homogeneous amplification (much longer in extent in F3) with concomitant copy number loss telomeric to the amplification. This pattern of amplification with distal loss is frequently observed in breast cancers on 8p [19]. Clone E8 showed a different pattern of amplification involving almost the entire 8p chromosome arm. Focal regions of copy number gain were interspersed with normal copy number in a sawtooth pattern, which has also been commonly observed in human tumors. Thus, these experimentally engineered multi-gene amplifications recapitulate several of the features of amplifications from actual human tumors. Importantly, the amplified subclones did not differ from parental MCF-7 or pre-amplified 38C-3 cells on the long arm of chromosome 8 (where MCF-7 has existing copy number gains, Fig. 3) or on the other chromosomes (not shown). This indicates that the induced copy number changes are specific and that the drug treatment does not select for generalized chromosomal instability.Fig. 3


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)

Copy number profile of chromosome 8 by array CGH. From top to bottom, MCF-7, the targeted, non-amplified MCF-7 clone 38C-3, and the mizoribine-amplified clones E8, F3, and G5. The y-axis represents log2 ratios of copy number, with 0 representing diploid copy number. Red boxes copy number gain, Green boxes copy number loss. The ZNF703 locus is indicated by an arrow. Copy number profiles did not differ from parental MCF-7 cells for the remaining chromosomes (not shown)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491111&req=5

Fig3: Copy number profile of chromosome 8 by array CGH. From top to bottom, MCF-7, the targeted, non-amplified MCF-7 clone 38C-3, and the mizoribine-amplified clones E8, F3, and G5. The y-axis represents log2 ratios of copy number, with 0 representing diploid copy number. Red boxes copy number gain, Green boxes copy number loss. The ZNF703 locus is indicated by an arrow. Copy number profiles did not differ from parental MCF-7 cells for the remaining chromosomes (not shown)
Mentions: To determine the extent and pattern of amplification, we performed genome-wide array CGH on the pre-amplified cells and amplified subclones (Fig. 3 and Supplemental Figures 1–3). As expected from the ddPCR result, all three subclones showed increased copy number of the ZNF703 locus. However, all three clones showed unique patterns of copy number change at surrounding loci. Clones F3 and G5 showed broad, homogeneous amplification (much longer in extent in F3) with concomitant copy number loss telomeric to the amplification. This pattern of amplification with distal loss is frequently observed in breast cancers on 8p [19]. Clone E8 showed a different pattern of amplification involving almost the entire 8p chromosome arm. Focal regions of copy number gain were interspersed with normal copy number in a sawtooth pattern, which has also been commonly observed in human tumors. Thus, these experimentally engineered multi-gene amplifications recapitulate several of the features of amplifications from actual human tumors. Importantly, the amplified subclones did not differ from parental MCF-7 or pre-amplified 38C-3 cells on the long arm of chromosome 8 (where MCF-7 has existing copy number gains, Fig. 3) or on the other chromosomes (not shown). This indicates that the induced copy number changes are specific and that the drug treatment does not select for generalized chromosomal instability.Fig. 3

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