Genome-Wide Gene Amplification during Differentiation of Neural Progenitor Cells In Vitro

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

Origin of cancer: an information, energy and matter disease

Cells are open, highly ordered systems far away from equilibrium. For that reason, the first function of any cell is to prevent the permanent threat of disintegration described by thermo-dynamic laws and to preserve highly ordered cell characteristics like structures, cell cycle and metabolism. In that context, three basic categories play a central role - energy, matter and information. Every single of these three categories is equally important to the cell and depends on the others reciprocally. For that reason, we suggest that either energy loss (e.g. by disturbed mitochondria) or disturbance of information (e.g. mutations, aneuploidy) or changes in matter composition or exposition (e.g. micro-environmental changes, toxic agents) can irreversibly disturb molecular mechanisms leading to increased local entropy of cellular functions and structures. In terms of physics, changes to these normally highly ordered reaction probabilities lead to a biologically irreversibly, imbalanced but thermodynamically more stable state. This primary change independent of the initiator now provokes and drives a complex interplay between energy availability, matter exposition and increasing information disturbance depending on reactions that try to overcome or stabilize this intra-cellular, irreversible disorder described by entropy. Because the return to the original ordered state is not possible due to the thermodynamic reasons cells die, or persist in an meta-stable state and enter into a self-driven adaptive and evolutionary process that generates progressive, disordered cells resulting in a broad spectrum of progeny with different characteristics, and maybe one day one of these cells will show an autonomous and aggressive behavior – a cancer cell

PCR Expression Analysis Of the Estrogeninducible Gene Bcei in Gastrointestinal and Other Human Tumors

A polymerase chain reaction (PCR) assay was developed to test for tumor cell specific expression of the BCEI gene. This new marker gene, reported at first for human breast cancer, was found specifically active in various gastrointestinal carcinomas by previously applying immunohistochemistry and RNA (Northern blot) analysis. Presently, by using reverse transcription -PCR analysis, a series of primary tumor tissues and established tumor cell lines were testcd for BCEI transcription. This approach was compared to immunostaining achieved by an antibody directed against the BCEI gene’s product. The result demonstrate the superior sensitivity of PCR by indicating the gene’ s expression in cases where immunohistochemical testing remained negative

Overview on amplified chromosomal regions.

<p>Chromosome regions that overlap with gained chromosome regions of TCGA glioblastoma samples are indicated in bold. Likewise, genes that were used for FISH analysis are indicated in bold. Examples of glioblastoma-amplified genes were included for chromosomal regions amplified after 5 d of differentiation. Start and end point were according to NCBI36/HG18.</p

Correlation of gene content to chromosome region.

<p>The correlation is shown for human chromosome 16 with several amplified and one deleted region in NHNP cells after 5 d differentiation. The log₂ ratio profile of the 10× window averaged data is presented at 25 kb resolution (A). Fused lasso analysis is performed using the same data points (B). GC repeats, gene content and banding pattern is shown as indicated by Ensembl genome browser (C). Both the log₂ ratio profile and the fused lasso analysis indicate an overlap between amplifications and regions with high GC and gene content and an overlap between deletion and regions with low GC and gene content.</p

Whole chromosome plots.

<p>A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.</p

FISH and immunofluorescence analysis.

<p>FISH with <i>GINS2</i> specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed <i>GINS2</i> amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with <i>CDK4</i> specific BAC (pink) and immunofluorescence staining with GFAP revealed <i>CDK4</i> amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with <i>CDK4</i> specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed <i>CDK4</i> amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.</p

FISH analysis of amplified loci.

<p>For each FISH analysis, a BAC or cosmid clone containing the indicated gene was Cy3-labelled (pink) and hybridized against fixed NHNP cells that were differentiated for either 2, 5 or 7 days. Amplifications are shown for <i>C1QL1</i> RP11-113A24 (5 days), <i>SOX13</i> RP11876H8 (5 days), <i>HDGF</i> RP11-66D17 (5 days), <i>CYP27B1</i> cosmid (2 days), <i>GINS2</i> RP11-118F19 (2 days), <i>CDK4</i> RP11-571M6 (2 days), <i>TP53</i> RP11-1081A10 (7days), <i>DIABLO</i> RP11-568C23 (7 days). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm. Notably, the degree of amplification various within each analysis due to the high heterogeneity of the amplifications in each cell population.</p

Detailed gene amplification analysis on human chromosome 16 and 12.

<p>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 <i>GINS2</i> specific BAC probe that was labeled in pink and a <i>CDH13</i> specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. <i>GINS2</i> amplification is indicated as pink speckled fluorescence signals whereas the neighboring <i>CDH13</i> gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without <i>GINS2</i> amplification are shown in Figure Bii. A <i>CDK4</i> specific BAC probe that was labeled in pink and a <i>XRCC6BP1/KUB3</i> specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. <i>CDK4</i> and <i>KUB3</i> amplifications were detectable as cluster of pink and green speckled fluorescence signals. <i>CDK4</i> specific signals spread over a more extended area than the <i>KUB3</i> specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.</p