Chapter 7 Chapter 7 Brakes: The Discovery of Tumor Suppressor Genes

In 1982, the discovery of gene point mutations was of great attraction to molecular biologists.Their consistent goal is to reduce the complex biological mechanisms of tumors to simple basic mechanisms.For cancer to develop, one mutation in the genome of a normal cell is enough.They like the view.But that same year, with the discovery of partner oncogenes, the number of mutations necessary to form a tumor climbed to two.But even that number has fascination for molecular biologists.The complexity represented by the two mutant genes is still manageable.Yet this figure is still under fire.By the mid-1980s, it became increasingly apparent that most tumors must accumulate far more than two mutations during their development.Data from epidemiology suggest that cancer requires at least six steps; many scientists speculate that each of these steps represents the creation of a new mutated gene on the way to the abyss of cancer.

This realization prompted a search for multiple mutated oncogenes predicted to be present in the genomes of human cancer cells.The researchers who conducted the manhunt were met with surprise and deep disappointment.They could not find populations of mutated oncogenes that co-existed in the genome of one tumor cell.Some tumors have the CC oncogene, others myc or NyC or erb, B2 but even two oncogenes are extremely rare.The idea that the development of cancer is the continuous activation of a set of oncogenes collapsed.Something must be terribly wrong. There are two ways out of the predicament.Or, contrary to much circumstantial evidence, perhaps tumors do not harbor multiple mutated genes.Either that or cancer cells do have six or more mutated genes, most of which have nothing to do with oncogenes.These putative genes may play an equally pivotal role in the development of human tumors.If so, genetic researchers are looking for a fish in the woods.They blinded their eyes and did not see Mount Tai. They took oncogenes as all oncogenes, and made a directional error.

By the mid-1980s, a mutant gene that was very different from an oncogene was finally found in human tumor DNA.The newcomers have been dubbed "tumor suppressor genes."The discovery closes a major hole in the mystery of human tumor formation.The discovery of this new gene type was carried out in experiments far removed from the studies of viruses, gene cloning, and gene transplantation that led to a surge of interest in oncogenes in the decade after 1975. The researchers used a fancy experimental procedure called "cell hybridization."Henry Harris at the University of Oxford has mastered this technique by allowing groups of cells to live on the bottom of petri dishes and making them fuse with each other.Through this fusion—that is, mating between cells—Harris and his followers discovered the truth about the behavior of genes inside cancer cells, including the discovery of tumor suppressor genes.

Long before cell fusion experiments began in the mid-1970s, geneticists were already performing mating experiments between organisms.As mentioned earlier, in the 1860s, Gregor Mendel, the Austrian soil repairer, conducted the first systematic genetic mating research. He did hybridization experiments on different strains of Pisum plants.His work fell into obscurity for a generation and was not seen again until 1900.The laws of inheritance he discovered laid the foundations of modern genetics, leading to the idea that biological information is conveyed by distinct packets of information, later called genes.

The breakthroughs in genetics in the 20th century revealed that all organisms, including the simplest single-celled organisms such as bacteria and yeast, use genes as templates for reproduction.And virtually all organisms, from bacteria to humans, have evolved elaborate mating mechanisms.The underlying motivation is the same across species, and it's obvious: mating enables the exchange and fusion of genes among species members.Because all species are populations of genetically diverse individuals, mating provides the opportunity to test new combinations of genes.New combinations of genes have the potential to produce offspring that are better suited to survival than their parents.In turn, this increasing fitness opens the way for evolution.

Controlling the mating of individuals with different genetic characteristics has become a powerful tool for studying the behavior of genes—especially how the genes of one member of the mating fuse with the genes of the other.Bacteria and yeast are able to mate with each other, whereas mammalian tissue cells lack this ability.The only natural mating between mammalian cells is the fusion of sperm and egg.This fact prevents researchers from seeing the results of mating different types of cells, such as bone cells from one person with bone cells from another, or bone cells with muscle cells from the same person.

Harris tries to circumvent the constraints imposed by nature.First, he forced animal cells in a dish to fuse with each other.The fusion of these cells, although highly artificial, provides a way for cells of different origin to mate with each other.Harris' fusion technique relies on the ability of certain virus particles to fuse the outer membranes of one cell with another adjacent cell in a petri dish.The result is that the nuclei of the two mother cells share an outer membrane.Soon, the two nuclei also fuse, their genes fused into a single nucleus. Under certain conditions, dozens of cells may be involved in a confluent vortex at the same time, forming cell giants too unwieldy to grow and divide.But fusion between two cells is much more interesting.The hybrid offspring of a cell pair can grow, divide, and pass on genes from both parent cells to their offspring.

As with most marriages, the mating of two cells is only interesting if the spouses are very different.People are interested in genetics because people want to predict the characteristics of offspring.Which side's genes are more influential? Human genetics begs a similar question: Will Little John's eyes look like his father's or his mother's?Will he have red hair like his father or brown hair like his mother? The results are often unpredictable, suggesting that the genes provided by each parent are at war.In whole organisms, whether yeast or humans, mating offspring have two copies of a gene that serves as a template for a particular shape.The signals from the two copies of the gene may be contradictory.John may have inherited a gene for brown eyes and a gene for blue eyes from his parents.The question is which gene will ultimately determine the color of his eyes.

Winners are often called dominant genes, and losers are recessive genes.Dominant genes are often more authoritative in influencing a cell's metabolism.For example, a dominant gene for eye color might have the ability to synthesize eye pigment, while a recessive gene would not. Based on the above considerations, Harris performed fusion experiments on human and rodent cells in different combinations, trying to figure out how their genes are integrated into one.The most exciting is the arranged marriage of normal cells and cancer cells.He will mix the cells together in a dish, pair them up to fuse, and study how the offspring of normal and cancer cells behave.

The results of cell hybridization seemed to be clear.Cancer is a dominant factor in living organisms, and the growth of tumor cells is undoubtedly stronger than that of normal cells.Therefore, if cancer cells and normal cells are fused, the cancer cell's vigorous genes will definitely show an overwhelming advantage.According to this reasoning, although the hybrid cell has two sets of genes, it must behave like the parental cancer cell.Injecting the hybrid cells into mice or rats should seed tumors. But Harris found the exact opposite.Hybrids of normal cells and cancer cells never have the ability to cause tumors.The prevailing prediction made a big mistake of 180". The growth genes of normal cells are dominant; the cancer-causing genes are recessive.

There is only one plausible explanation for Harris' bizarre conclusions. Normal cells seem to have genes that regulate normal cell growth.Instead, tumor cells must have discarded these genes on their way to becoming cancerous, and thus are not affected by the normal growth properties of these genes.After Harris arranges two cell bridal candles in the petri dish, the normalizing gene provided by the normal cell side regains control over the cancer cells, forcing the prodigal son of cell growth to turn back and go on the right track. This kind of thinking that the height of the devil is one foot, and the height of the Tao can be further deepened.Genes in normal cells seem to be slowing the cells down.In fact, they act as brakes, enabling cells to resist their tendency to overgrow.Since cancer cells have lost these genes, they have no brakes.Once cell hybridization reinstalled the brakes on the cancer cell, its rampant growth was stopped.Now, finally, the galloping horse—the infinite growth tendency of cancer cells—can be put on the bridle. None of the above findings agree with the prevailing belief that oncogenes are dominant.The latter is the culmination of the researchers' 10-year study of cancer genes.When an oncogene activated by a mutation is injected into a cell carrying a normal proto-oncogene, the oncogene should undoubtedly dominate.They enslave normal genes, forcing cells to grow out of control.That is to say, the proto-oncogene, as a recessive gene copy, has the function of promoting the step-by-step and normal reproduction of cells; while the mutant oncogene is a hyperactive dominant expression form, which is the driving force for the unremitting proliferation of cancer cells. Therefore, since Harris' growth-normalizing gene has a very different function from proto-oncogenes and oncogenes, it is necessary to give it a new name.According to its cell fusion · 48 · Combined expression, it is called a tumor suppressor gene.Both hyperactive dominant forms of proto-oncogenes and indolent, cryptically expressed tumor suppressor genes appear to play well in cancer development. Although it will take several years to isolate tumor suppressor genes through gene cloning technology, there is strong evidence that all researchers trying to understand the genetic basis of cancer cannot ignore the existence of tumor suppressor genes. So far, two sets of genetic actors have emerged on the cancer scene, each playing a different role in the mechanisms that control cell growth.A proto-oncogene is like the gas pedal of a car, and its mutant oncogene form is like stepping on the gas pedal to the bottom.In contrast, tumor suppressor genes function like cars, and when normal cells develop into cancerous cells, they may jettison or inactivate tumor suppressor genes, resulting in a defect in the brake mechanism.Either of these mechanisms seems to explain how the cell overgrowth works. There are two very different explanations for the formation of cancer, and there should be a trade-off.Could it be that some tumor cells use one mechanism to achieve cancerous growth while others use another?Or are there two mechanisms operating at the same time inside cancer cells?Perhaps, a combination of stomping the gas pedal and malfunctioning brakes contributed to the cancer's development. Mountains and rivers are full of doubts, and there is no way out, and the answer will not come so quickly.But the discovery of tumor suppressor genes did open another door in cancer research -- the heritability of cancer.Cancer often has a family history, and tumor suppressor genes provide a rationale for the origin of many familial cancers.cancer in the eye Harris' research shows that the loss of tumor suppressor genes plays a key role in the development and progression of certain cancers.Once the cell sheds the gene's repression, the cell's growth program kicks into gear.If the brakes fail, the car will inevitably lose control like a wild horse. Cells can inactivate or discard genes in many ways.Almost all methods are accomplished via mutations in the DNA sequences that make up genes.There is often a large segment of DNA base chain missing in the middle of the gene.Occasionally, a region of a chromosome that contains many genes is discarded. But the easiest, and therefore most common, way for a cell to remove a gene is much more subtle.A change of just one base in the base sequence of a gene—a point mutation—is most common.Although subtle, the change can have fatal consequences if it occurs in a key sequence of the gene.Point mutations can insert certain inappropriate punctuation marks in a gene; since these punctuation marks usually mark the end of a gene, they can cause premature interruption of gene reading, causing the synthesis of proteins directed by that gene to shrink.In addition, the gene's protein product may have some sort of change in its chain of amino acids that causes that protein to malfunction.Regardless of the size of the mutation, the result is the same: the cell will be unserved by the mutated gene. In fact, the process of losing tumor suppressor genes is more complicated than that described above.Almost all human cells have two copies of genes, one from each parent.In the case of tumor suppressor genes, having two copies of the gene provides the cell with double insurance.In case a cell accidentally loses a tumor suppressor gene, the tumor suppressor gene in the other copy can be an excellent backup player.Half a brake pad is almost always on par with a whole when it comes to slowing cell growth. This double insurance reflects the body's general approach to prevent cancer from forming.It is very rare for a cell to lose a tumor suppressor gene, and it is very unlikely that it will lose two at the same time.In particular, the loss of genes through mutational inactivation is usually only a one in a million probability in each cell generation.Thus, the probability of losing both copies of a gene per generation is one in a million squared.However, the actual risk is somewhat higher—more than one in a billion—due to complex genetic mechanisms, some of which are discussed below.Even so, the chances of a cell inadvertently missing two important growth-control genes are very small, creating a high barrier against cell overgrowth. The driving force behind two consecutive hits to knock out tumor suppressor genes is key to the formation of many types of tumors.We first became aware of this dynamic while studying a rare eye tumor, retinal glioma.This tumor only occurs in children under the age of six or seven, and the incidence rate is only one in 20,000.More than 500,000 people die from cancer in the United States every year, but there are only about 200 new cases of retinal glioma each year.The rare tumor appears to arise from embryonic retinal cells that normally must grow into photoreceptors -- rods and cones -- that respond to light by sensing light and sending electrical signals through the optic nerve to the brain. The disease can be divided into two categories.Children with sporadic retinal glioma have no close relatives who have had the disease.In familial cases, more than one member of several generations of the family often suffers from this otherwise rare disease. In 1971, Texas pediatrician Alfred Knudson proposed a theory that united the two types of retinal gliomas under one genetic umbrella.He believes that a retinal cell must undergo two genetic mutations to develop into a retinal glioma.In the sporadic form, a retinal cell undergoes two mutations in succession, either during embryonic development or shortly after birth, before it begins to grow out of control. Knudsen believes that the family type of the disease has two mutations.One mutation has already appeared in the fertilized egg that eventually develops into a baby.The mutation may have been inherited from a parent with the same disease, or it may have occurred during the formation of a sperm or egg and passed on to all the cells of the developing embryo.In turn, all cells of the newborn - most importantly including retinal cells - received a copy of the mutated gene.After that, any retinal cell needs only one more mutation to meet the double mutation conditions necessary to induce eye cancer. Recall the somatic mutations that hit the genomes of all cells except the gonads.Because mutations occur purely by chance, the possibility of two somatic mutations occurring in the same retinal cell is extremely small.In fact, only 1 in 4000 children has sporadic retinal glioma; and the number of retinal tumors in children is always only 1. In contrast, in the onset of familial retinal glioma, a single accidental somatic mutation is enough to trigger tumor growth.Due to the large number of target cells in the retina (more than 10 million) and the probability of a single cell mutation being one in a million, children who inherit the mutated gene and the associated predisposition to retinoid glioma often have multiple eyes in both eyes. a tumor occurs.In this case, every retinal cell is in fact at risk, and a single somatic mutation can make it go nowhere. By the middle of the 20th century, the situation with regard to mutations and genes affected by mutations had become clearer.The two target genes are two copies of a gene located on human chromosome 13, called the Rb gene because of its association with retinal glioma.Knudsen expects each mutation to knock out one copy of the Rb gene.When only one copy of the gene is inactivated, the retinal cell continues to grow completely normally with the spare copy of the gene.However, if both copies of the Rb gene are lost, the mechanism that controls cell reproduction is completely disrupted—the cell loses its brakes. All the characteristics of tumor suppressor genes predicted by the Harris cell fusion experiment are reflected in the Rb gene.The Rb gene exists in the normal cell genome, but the Rb gene in the tumor cell genome is either missing or functionally inactive.But now, new insights have emerged, building on Harris' earlier work.First, the loss of function of the tumor suppressor gene occurs in two steps, in which two copies of the gene are lost sequentially.Second, defective forms of tumor suppressor genes can be passed from parent to child via sperm or egg, resulting in an innate predisposition to tumors. The DNA sequence constituting the Rb gene was isolated by gene cloning in the joint efforts of my and Thaddeus Dryia's respective laboratories.Cloning allows us to fully estimate the role of the Rb gene in the origin of human cancer.At first glance, the role of the Rb gene appears to be limited to causing this rare childhood retinal tumor.In fact, however, the Rb gene appeared to be mutated in all of these tumors.In addition, children with a history of familial retinoid glioma in childhood are known to be at increased risk of bone cancer (osteosarcoma) during adolescence; loss of function of the Rb gene is also seen in these tumors. In the late 1980s, using the latest cloned Rb gene, it was revealed that more than one-third of bladder cancers and a small proportion (about 10%) of breast cancers also had loss of Rb genes, both of which were lost through the target organ. caused by somatic mutations.A genetic analysis of small-cell lung cancer, one of the most common causes of death among smokers, has revealed surprising results.All such tumors shed two copies of the Rb gene almost sequentially during their formation. We're starting to realize that the Rb gene actually plays a much broader role in the origin of cancer than we originally thought, that it was only associated with a rare childhood tumor.The long list of Rb-associated cancer classes led to a major question: What common traits link cells in these many different diseased organs throughout the body?The Rb gene of all cells in the body acts as a growth inhibitor. Why are these specific tissues particularly prone to cancer after loss of the Rb gene?The answer to the mystery may not be revealed until many years later.Loss of Diversity Now that we know of more than a dozen tumor suppressor genes, the Rb gene is just one of the top ones on the list.Finding these genes was not easy.Their presence is highlighted only when they are absent.How do you find the genes that are surreptitiously affecting cells behind the scenes?Some of these genes are associated with familial cancers such as retinal glioma; like the Rb gene, their mutated, defective forms can be transmitted through the germline pathway.Other tumor suppressor genes were not associated with congenital cancer susceptibility.Somatic mutations hit this or that target organ in situ, then sequentially wipe out both copies of the gene, knocking out the tumor suppressor gene.With clever tricks, we can track many of these genes.The success of the strategy depends on the specific genetic mechanism by which the two copies of the tumor suppressor gene are lost during tumor development.The most direct route is that the frequency of losing a copy of a gene in each generation of cells is one in a million.Then, another one-in-a-million mutation occurs in the same cell or one of its direct descendants, knocking out the other surviving copy of the gene.After losing both copies of the gene, the cells started growing out of control.As mentioned earlier, the probability of the same cell (or a small group of cells) undergoing two mutations is determined by the probability of each mutation occurring, which is about one in a trillion (one in a million squared) per cell generation ).Such a small probability means that in the normal human life cycle, such an event is extremely rare.When eliminating a second copy of a tumor suppressor gene, tumor cells often take a shortcut.Since the two partners in a human chromosome pair (such as chromosome 13, each with a copy of the Rb gene) always stand side by side in parallel formations, look at each other, compare their DNA sequences, and exchange genetic information .A common consequence is that a gene sequence in one chromosome replaces the corresponding sequence in the other.Before the transfer of information, each gene of a pair of chromosomes may have two different forms; after the transfer of information, one form is lost and replaced by a second copy of the gene originally present in the other chromosome.The result is that cells have two identical copies of genes that should be distinct. Loss of genetic diversity within a cell is often referred to as "loss of heterozygosity".The two copies of the gene show the same face - they assimilate. Assimilation of this or that gene occurs about 1 in 1,000 cell divisions.Thus, with this method, the other copy of the tumor suppressor gene that is intact can still be easily lost.That is, the good copy of the gene is discarded and replaced with a spare copy of the mutated, defective gene.The probability of gene assimilation is one in a million (the probability that the first copy of the gene is inactivated) times one in a thousand (duplicating the inactive gene, discarding the active gene), giving a probability of one in billion per cell generation . Precancerous tumor cells often use this strategy to eliminate two copies of a tumor suppressor gene that inhibits their growth as they become cancerous.They first undergo mutations that inactivate one copy of the tumor suppressor gene; then, through a process of assimilation through loss of heterozygosity, eliminate the second copy.Importantly, the exchange of chromosomal information leading to assimilation is not limited to the tumor suppressor gene, but often involves a large region of the chromosome surrounding the gene.Hundreds of genes on one chromosome, to the left and right of tumor suppressor genes, also assimilated. Of course, the assimilation of adjacent gene copies was not associated with the growth of developing tumor cells.They are nothing more than innocent bystanders implicated.The main enemy that tumor cells use assimilation strategy against is tumor suppressor gene. The fate of neighboring genes provides a breakthrough for geneticists trying to locate and isolate new tumor suppressor genes.Because of their loss of heterozygosity, one can analyze a large set of randomly selected genes scattered across the chromosomes of tumor cells.Genes that appear in two different forms in normal cell DNA but in the same form in his cancer cells are what geneticists are looking for.Regardless of the gene, any loss of diversity means that it is close in the chromosome to a tumor suppressor gene that is the true target of assimilation during tumor cell development. Following this logic, geneticists performed hundreds of searches in tumor cell genomes, looking for chromosomal regions that were repeatedly assimilated during tumor development.They suspected that these regions were hiding places for tumor suppressor genes.Once these regions are located, geneticists can use gene cloning techniques to find and isolate suspect molecules. So far, more than a dozen tumor-suppressor genes have been scooped up in the hunt by gene cloners.In nearly all colon cancers during their development, the chromosomal region near the A family gene is assimilated.During the development of neurofibromas, the region adjacent to the NF-1 gene loses diversity.The same fate was seen in the chromosomal region near WT-1 in some childhood renal cancers, and assimilation of the VHL region in adults with this disease.Loss of heterozygosity in the p16 gene can be seen in a variety of tumor development. This roster gives the impression that the human genome is rich in tumor suppressor genes.Three or four dozen were expected, but that number is too imprecise.Previous genetic discoveries led to the cloning of the Rb gene, and the discovery of so many genes this time has also led to a mystery that has not been solved until now: Although the vast majority of these genes can exist in many types of cells throughout the body, most of them are in the body. Loss only strongly affects the growth of some specific tissues, leaving other tissues unharmed. But certain genes remain unique relative to patterns that target specific tissues. The p53 tumor suppressor gene is well represented in a wide variety of cancers, with mutated forms of p53 present in as many as 60 percent of human cancers. Mutated forms of the p53 gene can also be passed from parent to child, resulting in a broad lifetime susceptibility to cancer and sarcoma. Finding new tumor suppressor genes remains arduous.The discovery of each gene requires many people to spend many years of hard work.After all, the discovery of loss of heterozygosity in the chromosomes of certain types of tumor cells is just a starting point for molecular hunters, who have to comb through millions of DNA bases to find a target tumor suppressor gene. The discovery process of new tumor suppressor genes has been greatly simplified due to the continuous progress of the Human Genome Project in the classification and mapping of human genes.What used to take years to find a single gene will be compressed to a few months in the near future, and many gaps in the genetic mystery of cancer will be filled.With these genes in hand, we can write a detailed development history of many tumors around the mutated oncogenes and tumor suppressor genes accumulated by tumors on the road to cancer.