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Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

Fischer U, Keller A, Voss M, Backes C, Welter C, Meese E - PLoS ONE (2012)

Bottom Line: We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells.Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells.Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Saarland University, Homburg/Saar, Germany. ulrike.fischer@uks.eu

ABSTRACT
DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

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Detailed gene amplification analysis on human chromosome 16 and 12.Representative sections of log2 ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log2 ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A GINS2 specific BAC probe that was labeled in pink and a CDH13 specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. GINS2 amplification is indicated as pink speckled fluorescence signals whereas the neighboring CDH13 gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without GINS2 amplification are shown in Figure Bii. A CDK4 specific BAC probe that was labeled in pink and a XRCC6BP1/KUB3 specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. CDK4 and KUB3 amplifications were detectable as cluster of pink and green speckled fluorescence signals. CDK4 specific signals spread over a more extended area than the KUB3 specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.
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pone-0037422-g003: Detailed gene amplification analysis on human chromosome 16 and 12.Representative sections of log2 ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log2 ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A GINS2 specific BAC probe that was labeled in pink and a CDH13 specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. GINS2 amplification is indicated as pink speckled fluorescence signals whereas the neighboring CDH13 gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without GINS2 amplification are shown in Figure Bii. A CDK4 specific BAC probe that was labeled in pink and a XRCC6BP1/KUB3 specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. CDK4 and KUB3 amplifications were detectable as cluster of pink and green speckled fluorescence signals. CDK4 specific signals spread over a more extended area than the KUB3 specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.

Mentions: As further validation step we compared the amplification event between genes from two neighboring chromosome regions. Within chromosome region 16q24.1 the log2 ratio values revealed an increase of genomic sequences at 83.7–84.4 Mb. In the same chromosome region, the log2 ratio values indicated a normal copy number at 82 Mb (Figure 3A). FISH experiments revealed amplification of the GINS2 gene that maps at 84.27 Mb, but no amplification for CDH13 at 82 MB (Figure 3Bi). FISH analysis also provided evidence for a large heterogeneity of amplifications in neighboring cells. Figure 3Bii shows a nucleus with amplified GINS2 fluorescence signals next to a nucleus with only 3 GINS2 specific fluorescence signals.


Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

Fischer U, Keller A, Voss M, Backes C, Welter C, Meese E - PLoS ONE (2012)

Detailed gene amplification analysis on human chromosome 16 and 12.Representative sections of log2 ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log2 ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A GINS2 specific BAC probe that was labeled in pink and a CDH13 specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. GINS2 amplification is indicated as pink speckled fluorescence signals whereas the neighboring CDH13 gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without GINS2 amplification are shown in Figure Bii. A CDK4 specific BAC probe that was labeled in pink and a XRCC6BP1/KUB3 specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. CDK4 and KUB3 amplifications were detectable as cluster of pink and green speckled fluorescence signals. CDK4 specific signals spread over a more extended area than the KUB3 specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351388&req=5

pone-0037422-g003: Detailed gene amplification analysis on human chromosome 16 and 12.Representative sections of log2 ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log2 ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A GINS2 specific BAC probe that was labeled in pink and a CDH13 specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. GINS2 amplification is indicated as pink speckled fluorescence signals whereas the neighboring CDH13 gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without GINS2 amplification are shown in Figure Bii. A CDK4 specific BAC probe that was labeled in pink and a XRCC6BP1/KUB3 specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. CDK4 and KUB3 amplifications were detectable as cluster of pink and green speckled fluorescence signals. CDK4 specific signals spread over a more extended area than the KUB3 specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.
Mentions: As further validation step we compared the amplification event between genes from two neighboring chromosome regions. Within chromosome region 16q24.1 the log2 ratio values revealed an increase of genomic sequences at 83.7–84.4 Mb. In the same chromosome region, the log2 ratio values indicated a normal copy number at 82 Mb (Figure 3A). FISH experiments revealed amplification of the GINS2 gene that maps at 84.27 Mb, but no amplification for CDH13 at 82 MB (Figure 3Bi). FISH analysis also provided evidence for a large heterogeneity of amplifications in neighboring cells. Figure 3Bii shows a nucleus with amplified GINS2 fluorescence signals next to a nucleus with only 3 GINS2 specific fluorescence signals.

Bottom Line: We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells.Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells.Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Saarland University, Homburg/Saar, Germany. ulrike.fischer@uks.eu

ABSTRACT
DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

Show MeSH
Related in: MedlinePlus