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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

Proteins in the amplified region are overexpressed. Western blot for selected proteins from the 8p11-12 region. Lane 1, MCF-7. Lane 2, MDA-MB-134VI, a human breast cancer cell line with known amplification of 8p11-12. Lane 3, the targeted, pre-amplified 38C-3 clone. Lanes 4–6, amplified subclones derived from 38C3. ZNF703, FGFR1, RAB11FIP1, and ASH2L are encoded by genes on 8p11-12. Asterisks indicate C-terminal proteolytic processed fragments of FGFR1. Migration of molecular weight standards in kilodaltons is indicated on the right
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Fig6: Proteins in the amplified region are overexpressed. Western blot for selected proteins from the 8p11-12 region. Lane 1, MCF-7. Lane 2, MDA-MB-134VI, a human breast cancer cell line with known amplification of 8p11-12. Lane 3, the targeted, pre-amplified 38C-3 clone. Lanes 4–6, amplified subclones derived from 38C3. ZNF703, FGFR1, RAB11FIP1, and ASH2L are encoded by genes on 8p11-12. Asterisks indicate C-terminal proteolytic processed fragments of FGFR1. Migration of molecular weight standards in kilodaltons is indicated on the right

Mentions: There have been few systematic investigations of overexpression of amplified genes at the protein level, although individual candidate genes have been studied and documented, such as HER-2. We examined protein expression for several of the genes in the region, compared to parental non-amplified MCF-7 cells and the 8p11-12 amplified breast cancer cell line MDA-MB-134VI (Fig. 6). We observed protein overexpression of full length FGFR1 or its proteolytically processed C-terminal fragments in clones F3 and G5 [27]. These clones also overexpressed RAB11FIP1, and F3 additionally overexpressed ASH2L. Protein expression differences for ZNF703 were less dramatic, in keeping with the low level increase in mRNA. It should be noted that the targeted IMPDH cassette is translated from an IRES, allowing independent posttranscriptional regulation of ZNF703. Thus, experimental amplification of a targeted locus can lead to overexpression of regional genes at the protein level, even when direct selection for the activity of these proteins is not applied.Fig. 6


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)

Proteins in the amplified region are overexpressed. Western blot for selected proteins from the 8p11-12 region. Lane 1, MCF-7. Lane 2, MDA-MB-134VI, a human breast cancer cell line with known amplification of 8p11-12. Lane 3, the targeted, pre-amplified 38C-3 clone. Lanes 4–6, amplified subclones derived from 38C3. ZNF703, FGFR1, RAB11FIP1, and ASH2L are encoded by genes on 8p11-12. Asterisks indicate C-terminal proteolytic processed fragments of FGFR1. Migration of molecular weight standards in kilodaltons is indicated on the right
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491111&req=5

Fig6: Proteins in the amplified region are overexpressed. Western blot for selected proteins from the 8p11-12 region. Lane 1, MCF-7. Lane 2, MDA-MB-134VI, a human breast cancer cell line with known amplification of 8p11-12. Lane 3, the targeted, pre-amplified 38C-3 clone. Lanes 4–6, amplified subclones derived from 38C3. ZNF703, FGFR1, RAB11FIP1, and ASH2L are encoded by genes on 8p11-12. Asterisks indicate C-terminal proteolytic processed fragments of FGFR1. Migration of molecular weight standards in kilodaltons is indicated on the right
Mentions: There have been few systematic investigations of overexpression of amplified genes at the protein level, although individual candidate genes have been studied and documented, such as HER-2. We examined protein expression for several of the genes in the region, compared to parental non-amplified MCF-7 cells and the 8p11-12 amplified breast cancer cell line MDA-MB-134VI (Fig. 6). We observed protein overexpression of full length FGFR1 or its proteolytically processed C-terminal fragments in clones F3 and G5 [27]. These clones also overexpressed RAB11FIP1, and F3 additionally overexpressed ASH2L. Protein expression differences for ZNF703 were less dramatic, in keeping with the low level increase in mRNA. It should be noted that the targeted IMPDH cassette is translated from an IRES, allowing independent posttranscriptional regulation of ZNF703. Thus, experimental amplification of a targeted locus can lead to overexpression of regional genes at the protein level, even when direct selection for the activity of these proteins is not applied.Fig. 6

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