Extrachromosomal DNA Single cell sequencing Cell Family Video


Where does DNA exist in eukaryotic cells? If you have studied high school biology, you should be able to answer easily: cell nucleus, mitochondria, chloroplast.

Most of the DNA is located in the nucleus. According to all current textbooks, in the nucleus of eukaryotic cells, DNA is wrapped around histones in circles to form chromosomes with high-level structures. So we have always had this basic understanding: nuclear DNA is all located in chromosomes.

However, the reality is not so perfect.

In special circumstances (excluded here is the invasion of foreign nucleic acids such as viral infections), nuclear DNA may be separated from the chromosomes. For example, due to factors such as ionizing radiation and ultraviolet rays, DNA double-strand breaks are caused. Under normal circumstances, our cells have a corresponding repair mechanism to make up for the missing DNA and discharge the spent DNA out of the cell.Family Video, Assuming that the damage is too great to be repaired, the cell will also activate the death mechanism to avoid expressing the wrong gene, and also avoid passing the wrong genetic information to the offspring cells.

However, there is a kind of maverick cell in this world. There is an error in their genetic information, which is not easy to repair, and they do not want to die, which makes us very embarrassed. And this type of cell is my research object-tumor cells.

Recently, our laboratory published an article about extrachromosomal DNA (ecDNA) of tumor cells in Nature : Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. (Porta Taking this opportunity, this column will publish 3 articles in a row to introduce readers to all aspects of tumor ecDNA.

1. Ploidy abnormalities and gene amplification are common features of malignant transformed cells

As early as 1842, Swiss botanist Carl Wilhelm von Nägeli was the first to observe the existence of chromosomes, which laid the foundation for cytogenetics. However, it was not until 1956, more than a hundred years later, that scientists formally determined that human somatic cells have 46 chromosomes.Family Video USA, However, before the formal determination of the number of chromosomes, there have been many studies dedicated to observing the chromosomes of human tumors, and have reached a consistent conclusion: the number of chromosomes of malignant transformed cells, or tumor cells, is inconsistent with that of normal cells, and is usually irregular. Ploidy increased. The figure below is a very early literature, reporting that human glioma has 81 chromosomes.

An increase in the number of chromosomes means an increase in the number of gene copies. If the number of genes that stimulate growth increases, then the proliferation of this cell will easily get out of control, and will evolve into a tumor driven by complex multi-gene interactions and environmental selection. For example, chromosome 7 in many epithelial tumors tends to increase, and this chromosome carries the EGFR gene (epidermal growth factor receptor) that drives this type of tumor .

Is the increase in the number of chromosomes the only mechanism for the increase in the copy number of tumor cells? Actually not. In the chaotic karyotype of tumor cells, there are actually many possibilities for gene amplification.

2. Extrachromosomal DNA carries amplified proto-oncogene

When studying the tumor karyotype, scientists discovered a very strange phenomenon. In addition to the aneuploidy increase in the number of chromosomes, it can also be observed: 1.F M TV, There are many small spots outside the chromosomes that can be colored by dyes; 2. When band dyeing, some chromosomes will be found to show large areas of bright or dark bands .

As shown in Figures A and B below, we can find that there are some large or small staining signals outside the chromosomes. Figure C shows that the chromosomes have unusually long bright bands.

What are the little dots in pictures A and B? Is it an impurity in the dye? Did you accidentally break the chromosome when preparing the sample? The answer is no, because when using the same method to prepare samples of normal cells, these small spots were not found, indicating that this is not caused by technical errors. And the large-area abnormal banding pattern in Figure C is not because the chromosomes are glued together when preparing the sample. After the development of fluorescence microscopy and fluorescence in situ hybridization (FISH), we finally understand that there are a large number of copies of proto-oncogenes.

For example, in the tumor cell line above, we use DAPI (blue) to stain DNA, red FISH probe to stain the proto-oncogene EGFR , and green FISH probe to label human chromosome 7. (Note: The EGFR gene is located on chromosome 7. And this picture is from our laboratory.)

It can be found that if only DAPI is used to label DNA, many small spots free of chromosomes, namely ecDNA, can also be observed in the sample on the left. It can be found by FISH probe that almost all ecDNA contains EGFR gene. In the sample on the right, the EGFR gene signals are “squeezed” together to form a strong staining area. This is also the reason why continuous large areas of bright or dark bands appear when doing karyotype staining. We call such staining regions homogeneously staining regions (HSR, uniformly staining regions). A phenomenon worth noting is that most of the EGFR gene on HSR is not on chromosome 7, but inserted into other chromosomes, or is self-contained.

3. ecDNA is ubiquitous in tumor cells

We can’t help asking, is the phenomenon of ecDNA unique to tumor cells? Is it the commonality of tumor cells? As shown in the figure below (the figures in Section 3 are from the Nature article, and the sources are not marked one by one), we have counted a total of 2572 metaphase metaphase karyotypes from 143 samples (only the metaphase karyotypes can be prepared Detection of ecDNA) found that ecDNA is rarely present in the metaphase of normal cells, the proportion of ecDNA in the metaphase of immortalized cells is increased, and the proportion of ecDNA in the metaphase of malignant transformed tumor cells is higher.

 This kind of graph is called beanplot, and the Chinese name is called bean pod plot for the time being. It is an upgraded version of the traditional box plot. It not only shows the distribution range of all data-it can display the maximum and minimum values, but also in the horizontal The relative distribution frequency of the data displayed in the direction-the “fat” the higher the relative frequency. As far as I know, the R language can be used to generate beanplots.)

If the tested metaphase is classified according to the type of cancer, we can see that the proportion and quantity of ecDNA in different tumor cell types are different. For example, the colon cancer cell line metaphase has a low ecDNA ratio and a small number, while the brain tumor GBM has a high ecDNA ratio and a large number.

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