Limits...
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-associated overexpression of co-amplified genes on 8p11-12. Quantitative real-time RT-PCR for selected genes in the 8p11-12 region in their genomic order (ZNF703, telomeric; MYST3, centromeric). Expression for each gene is normalized to a reference housekeeping gene, TBP. The expression level in the pre-amplified 38C-3 clone is set at 1. The mean and standard deviation of two experiments are represented
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4491111&req=5

Fig5: Copy number-associated overexpression of co-amplified genes on 8p11-12. Quantitative real-time RT-PCR for selected genes in the 8p11-12 region in their genomic order (ZNF703, telomeric; MYST3, centromeric). Expression for each gene is normalized to a reference housekeeping gene, TBP. The expression level in the pre-amplified 38C-3 clone is set at 1. The mean and standard deviation of two experiments are represented

Mentions: Copy number variation is a leading cause of gene expression variation among tumors, and copy number-associated overexpression can be used as a criterion to narrow down the list of candidate driver genes in a given region. We performed qRT-PCR for genes in the core 8p11-12 amplification in our experimentally amplified clones (Fig. 5). All clones showed increased expression of ZNF703, as would be predicted; however, the clones differed in the extent and degree of copy number-associated overexpression of neighboring genes in the region. Clone G5 showed the highest relative expression in the greatest number of genes, followed by clone F3, and clone E8 exhibited more modest changes. This trend is in keeping with the broader increase of copy number for these genes in G5 and F3 versus E8 seen by array CGH. These differences may also reflect epigenetic variation among the clones. Indeed, the correlation between copy number gain and gene overexpression in cancer-associated amplifications is imperfect.Fig. 5


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-associated overexpression of co-amplified genes on 8p11-12. Quantitative real-time RT-PCR for selected genes in the 8p11-12 region in their genomic order (ZNF703, telomeric; MYST3, centromeric). Expression for each gene is normalized to a reference housekeeping gene, TBP. The expression level in the pre-amplified 38C-3 clone is set at 1. The mean and standard deviation of two experiments are represented
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4491111&req=5

Fig5: Copy number-associated overexpression of co-amplified genes on 8p11-12. Quantitative real-time RT-PCR for selected genes in the 8p11-12 region in their genomic order (ZNF703, telomeric; MYST3, centromeric). Expression for each gene is normalized to a reference housekeeping gene, TBP. The expression level in the pre-amplified 38C-3 clone is set at 1. The mean and standard deviation of two experiments are represented
Mentions: Copy number variation is a leading cause of gene expression variation among tumors, and copy number-associated overexpression can be used as a criterion to narrow down the list of candidate driver genes in a given region. We performed qRT-PCR for genes in the core 8p11-12 amplification in our experimentally amplified clones (Fig. 5). All clones showed increased expression of ZNF703, as would be predicted; however, the clones differed in the extent and degree of copy number-associated overexpression of neighboring genes in the region. Clone G5 showed the highest relative expression in the greatest number of genes, followed by clone F3, and clone E8 exhibited more modest changes. This trend is in keeping with the broader increase of copy number for these genes in G5 and F3 versus E8 seen by array CGH. These differences may also reflect epigenetic variation among the clones. Indeed, the correlation between copy number gain and gene overexpression in cancer-associated amplifications is imperfect.Fig. 5

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