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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

Droplet digital PCR measurement of copy number at the ZNF703 locus at 8p12. Copy number is normalized to the two copy RPP30 locus. From left to right, parental MCF-7 cells, a targeted clone (38C-3) before mizoribine amplification selection, and three amplified subclones of the 38C-3-targeted clone (E8, F3, G5). Bars represent 95 % confidence intervals. Results are representative of three experiments
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Fig2: Droplet digital PCR measurement of copy number at the ZNF703 locus at 8p12. Copy number is normalized to the two copy RPP30 locus. From left to right, parental MCF-7 cells, a targeted clone (38C-3) before mizoribine amplification selection, and three amplified subclones of the 38C-3-targeted clone (E8, F3, G5). Bars represent 95 % confidence intervals. Results are representative of three experiments

Mentions: We designed homology arms to target the E. coli IMPDH cassette to the 3′ UTR of the ZNF703 gene, which is at the telomeric end of the core 8p11-12 amplicon (Fig. 1). Multiple targeted clones were identified by PCR screening and purified to homogeneity by limiting dilution. We next plated one of the targeted clones, named 38C-3, in mizoribine. We identified three colonies resistant to 10 μM mizoribine, designated as E8, F3, and G5. We initially tested these colonies for increased copy number of the targeted ZNF703 locus by performing qPCR with primers specific to the targeting cassette and to the ZNF703 locus outside of the region of the homology arms (data not shown). Subsequently, we used droplet digital PCR to more precisely measure copy number at the ZNF703 locus using primers and a probe located near exon 1, normalized to the RPP30 gene, of which MCF-7 has two copies. As shown in Fig. 2, clones E8, F3, and G5 showed average ZNF703 copy number increases of approximately 2.5-fold relative to parental MCF-7 and the targeted clone 38C-3 before mizoribine selection. This indicates that the ZNF703 amplification occurred during mizoribine selection and was not present in the targeted 38C-3 clone prior to selection.Fig. 2


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)

Droplet digital PCR measurement of copy number at the ZNF703 locus at 8p12. Copy number is normalized to the two copy RPP30 locus. From left to right, parental MCF-7 cells, a targeted clone (38C-3) before mizoribine amplification selection, and three amplified subclones of the 38C-3-targeted clone (E8, F3, G5). Bars represent 95 % confidence intervals. Results are representative of three experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491111&req=5

Fig2: Droplet digital PCR measurement of copy number at the ZNF703 locus at 8p12. Copy number is normalized to the two copy RPP30 locus. From left to right, parental MCF-7 cells, a targeted clone (38C-3) before mizoribine amplification selection, and three amplified subclones of the 38C-3-targeted clone (E8, F3, G5). Bars represent 95 % confidence intervals. Results are representative of three experiments
Mentions: We designed homology arms to target the E. coli IMPDH cassette to the 3′ UTR of the ZNF703 gene, which is at the telomeric end of the core 8p11-12 amplicon (Fig. 1). Multiple targeted clones were identified by PCR screening and purified to homogeneity by limiting dilution. We next plated one of the targeted clones, named 38C-3, in mizoribine. We identified three colonies resistant to 10 μM mizoribine, designated as E8, F3, and G5. We initially tested these colonies for increased copy number of the targeted ZNF703 locus by performing qPCR with primers specific to the targeting cassette and to the ZNF703 locus outside of the region of the homology arms (data not shown). Subsequently, we used droplet digital PCR to more precisely measure copy number at the ZNF703 locus using primers and a probe located near exon 1, normalized to the RPP30 gene, of which MCF-7 has two copies. As shown in Fig. 2, clones E8, F3, and G5 showed average ZNF703 copy number increases of approximately 2.5-fold relative to parental MCF-7 and the targeted clone 38C-3 before mizoribine selection. This indicates that the ZNF703 amplification occurred during mizoribine selection and was not present in the targeted 38C-3 clone prior to selection.Fig. 2

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