 It's on cancer. How does cancer rise? You may have heard that the prevailing theories that it's caused by a number of mutations, so what, mutation theory, but we have studying it run into more and more difficulties, was it, and come up with essentially a new old theory that is actually chaotic, very much like the evolution of species, exactly like the evolution of species almost, except that it doesn't make it all the way to autonomy like we and the other species that Darwin or God or somebody in between has created. And we don't know how. This is not so clear. With cancer we do know how. We can actually test it. It's a chromosomal evolution process. So this is what I'm going to talk about today. Now briefly, for those of you who are, all of you who are in this, the cancers contain individual clonal karyotypes, much like normal phyllo-genetic species. They're not a normal karyotype with mutation, as in mutation theory such as they have their own karyotypes with their own chromosomal numbers, everything. The karyotypes of cancer differ from normal cells, diploid cells, in cancer specific, abnormal, as you would call relative to the normal cell. Or aneuploid is the technical term, meaning it's a creek, whatever derivative term, not the right ploidy, not the right folding, but that number of chromosomes and structures. And here are two examples. On the left is the pinnacle of evolution. That's the human male with 23 chromosomes. Here diploid chromosomes. And this is by so-called M-fish. You color code these chromosomes. It makes it much easier to do side of genetic analysis. You spread the chromosomes on an object, on a microscope slide, and then hybridizing with color-specific probes. And they all assume these artificial colors. So this is chromosome one, two, three, all the way to 22. And here's X and Y, one each. So that's a normal human male. 46 chromosomes. And here is one particular cancer cell, a karyotype of a colon cancer cell line. It's very stable, in fact, not quite as stable as normal karyotypes are. But you can see the numbers are drastically changed. And here's a gallery of so-called creeps, almost, so-called marker chromosomes that are hybrids of these chromosomes. You can tell from the colors. This is a hybrid of 6 and 1. This is a hybrid of 13. Some 13 marker. This is when the two, with two colors, you could say 17, 22, and 17. The triple hybrids markers, that is the hallmark of cancer cells. When that happens in us normally, then things are very bad. You would hardly ever be born with any of these changes, only as a cancer cell are you born, which is sort of a new species. Here are two more examples. That is a breast cancer, not a good one, as you can see, lots of marker chromosomes, abnormal cells, breast cancer line. And as I said, everybody's cancer is individual. They all have their own karyotypes. Again, like species. And here's a karyotype of a muscle cancer that is simpler than the two others that are highly, more highly developed. So in contrast to the karyotypes of normal species, you can already see from these patterns, the karyotypes and phenotypes of cancers are flexible. I don't like to use the word unstable, because unstable means they would fall apart. But they oscillate in a narrow range, like radiation wave in physics. And they are flexible in these limits. And that's able to evolve ever more like phenotypes spontaneously. That is the daunting property of cancers. It starts bad, but it typically gets worse, because it's so flexible the evolution occurs very fast compared to Darwinian evolution. You have to wait for every step 10 million years. This explains the daunting ability of cancers to go from bad to worse, as Peyton Rouse used to call it, which is also termed now cancer progression. You start off with a manageable cancer that gets more and more drug-assistant, metastatic, and unpredictable. As a result of this inherent inflexibility, the karyotypes of cancers are typically also heterogeneous, and so are the phenotypes. But again, within limits. Now, this is a different way of looking at the instability. It's sort of a thing that is in Excel, a student of mine found, figured it out. You can line up three-dimensionally the metaphases or the chromosomes of 20 cells in the x-axis, and they call it the karyoglyph, the x-axis here. Down there are the chromosome numbers of a given cell. So here's chromosome one, two, three, four, five, and six. And this, you can see already, is a male cell, a normal male cell. We have two. Here's the y-axis is the chromosome copy number. There are two, because it's diploid. And in men, there's one only. It's haploid, if you like, monosomic of the x- and y-chromosomes. There are two of those here. All others are double. So these are images or essentially different cells. They're 20 usually lined up, and next to each other. And you can see three dimensionally that they're completely parallel, meaning that the karyotypes are identical and very stable. If you look with the same instrument, with the same diagram, at a cancer cell, this one is a breast cancer, another breast cancer line, you can see now that it is a completely different karyotype graphically. It isn't no longer the smooth parallel lines of a normal cell. They go up and down. They have a new karyotype. You see the copy number here of the maturity is three, then it goes down to one. Here it's up to four. Here it's even five. And you can see the lines are no longer totally parallel, but they oscillate around this pattern. And this pattern is actually remarkably stable. It's a stability within instability. That is characteristic of the cancer. Even if you passage this cancer 60 generations in culture or put it through an animal and isolate it again, it will look more or less the same, unless you put on some strong selective pressure like carc selection. So then it changes the karyotype, which the cancer cell can do. Normal cell never does. When you have chemotherapy, it's the cancer that becomes resistant and you never do. So as a result of this inflexibility, the karyotypes of cancer and the resulting tumors are heterogeneous and it is therefore still debated and has been debated for nearly 100 years now ever since for very first suggested chromosomal relations like causes of cancer. And subsequently, the majority of geneticists favored the view that it's mutations. It is still debated because they're flexible, but rather specific karyotypes, as I just showed to you, or whether stable and specific mutations are causes of cancer, hidden that we don't see or the so-called oncogenes now, that would, as a consequence of their presence, cause cancer and also destabilize the karyotype. So the destabilized karyotype is recognized even by the mutation people, although you can hardly find it in modern textbooks anymore. You don't see a karyotype anymore. Everything is expressed in genes or in gene arrays. The karyotype is considered something for little old ladies or people of the last generation are looking for microscopes and work on a low budget. But for the real advanced cytogenetic cancer researcher, you have a gene array for $1,000 apiece and show a diagram with thousands of little spots to interpret for the surgeon whether the right breast or the left breast or lymph node should be taken out or not. That's what they really say at the end. I don't know how they see it, but they do see a lot, I guess. So this is still the question that has not been settled in 100 years of debate. Is it chromosomes or is it mutations that are causing cancer? And we are solving it right now. Next 15 minutes, that's what we have. No more. So the mutation theory holds that a set of three to six mutations of specific genes that are termed oncogenes as a result of these mutations, namely, meaning that they cause cancer, transform normal cells to cancer cells independent of carrier-typic alterations. They recognize grudgingly, yes, that a lot of carry-type changes, but they say this is a consequence or an accidental thing because the cell is transformed. Popular proponents of the mutation theory, all of my former friends and even current friends have some of them like Bishop and Varmas and Weinberg and Vogelstein, so all the leading cancer researchers still subscribe to the mutation theory. So, but there is no direct functional proof for the mutation theory. Despite 30 years of research, and despite gene technology, there's marbles, you can put any gene in any cell you want, if you want. And they have done that with these oncogenes. They put them into normal cells, but as you can see, the results are at best ambiguous, if not, certainly no direct proof. What happened this when you take a convenient or whatever they call it, a consensus number of oncogenes that is said to be or sought to be sufficient for cancer, one of them published in Nature recently by Weinberg and by other Bishop and so on have done the same thing. They take three or four, put them into normal human cells, and then they say, see, when you wait long enough, after several months, there is a tumor cell coming up as a clone. What they don't tell you is they put millions of cells into this culture that all have the same oncogenes and are not transformed, out of which over months comes occasionally a clone of transformed cells. So, what that means is when you get for in for transfecting as that is called millions of cells with oncogenes that are supposedly causing the cancer, you get only one in ten to the five to transform, that means these genes are not sufficient to do the job, something else is required. And that is more or less openly acknowledged. So they are not sufficient for tumor genicity. And the answer of the mainstream is we need another gene yet that we haven't found yet. But they have never found a complete set yet, although the Nobel Prize was given for this idea already in 89. So have to catch up soon to justify the Nobel Prize or the next one, whatever it is. So the oncogenes that induce transformation is another result of these experiments. In one of ten to the five cells are not necessary to maintain the transformed phenotype. They do their job transiently because tumors and leukemias induced by oncogenes persist. Even if the oncogenes are lost or turned off experimentally with experimentally controllable promoters, these teton, tetof, you take tetracycline promoters and you give the mouse tetracycline and the gene is shut off or shut on. Once the tumor is established in a couple of weeks, it is no longer dependent on the oncogene. The oncogene, in other words, is not maintaining the transformed phenotype and it's not sufficient to initiate it. And all cells that have been studied for karyotypes, and that's only very few who have done that, you can imagine one of who did, who have found to be a duplicate. And that is not mentioned in nature when that was one of the leading papers when Weinberg came out. I even sent him the karyotypes. Isn't that interesting information at least? They said, no, no, we don't need that. It's enough that we have in there six mutations, that they have 70 chromosomes in these cells plus the six mutations that is equivalent of another roughly 20,000 genes. It's not relevant to them because they don't have any known mutations in them. So they're not taking this very serious. So what is now the role of mutations or oncogenes in cancer? The conclusions from these three kinds of experiments are oncogenes are not sufficient and are not necessary to maintain the cancer cells. So what is their role in transformation? That is the one experiment I wanted to describe today and then I will shut up. Here's the theory that we think how it works and what it does and the effect of cancer is generated in general. In efforts to explain this, we have recently proposed a two-step karyotypic cancer theory. That's once you have seen that, then it's almost, I'm almost over. The first step in this process of carcinogenesis is that carcinogens and also the oncogenes which function like carcinogens destabilize the karyotype by inducing random aneuploidy. Some of them might also induce mutations, but that's secondary. The critical essential point for cancer is that they destabilize the karyotype and that you see in every cancer cell and that explains at the same stroke about half of all known carcinogens that are not mutagens like asbestos and polystyrene and all these things. So once you have an aneuploid cell, the aneuploidy destabilizes the karyotype automatically, autocatalytically by unbalancing teams of proteins that segregate and synthesize and repair chromosomes. Imagine you had the most balanced and physically complex apparatus, a spindle apparatus in the cell, segregating 46 chromosomes symmetrically every eight hours if it has to be. And that works 99.9% accurate. If there's a slightest imbalance, if there's a little bit more of tupelein instead of clopylein or what have you, or myosin or something, then this thing is out of balance and it's like you have a long leg and a short leg. You start limping and you become aeroplane and the aneuploid karyotype constantly changes itself because of these many imbalances, physical imbalances in the spindle apparatus, which is a very balanced sensitive machine, or even in the enzyme teams, the repair enzymes, for example, that fix the DNA. If you ever done NIC translation, there's a DNAs, polymerase, and a ligase in a mix. If you change that balance a little bit, it's either hydrolyzing the DNA or it stops making it. If they are in the right balance, you get optimal yield and that balance is maintained by the balance of genes in the chromosomes that have been balanced for three billion years of life. Now, if you change that, if you change thousands of genes by aneuploidy, thousands of things are out of balance. In most cases, in fact, the aneuploid cell dies. It has to find an equilibrium that it can maintain to survive. So the aneuploidy stabilizes the karyotype autocatalytically and thus keeps changing the karyotype and thus initiates and maintains karyotypic evolutions. They're constantly recouped and changed because of the inherent instability. Most of these newly evolving karyotypes are again random aneuploidies that are functionally inferior to normal cells or even lethal and they're gone. Occasionally, however, like playing roulette and playing darbin, a real cancer-causing karyotype evolves. It's a long way from a real autonomous species that says goodbye mom and climbing up on the tree or flying like a bird. That's not it yet, but it's good enough to be an autonomous parasite. It can compete with normal cells from which it just arose. These cancer-causing karyotypes are then stabilized by the inherent instability against the inherent instability of aneuploidy by selection for transforming function. So they keep, as you could see on these karyocrafts that I showed earlier, they keep fluctuating or oscillating around clonal value, but as soon as they go too far outside it, they will not replicate and not be maintained as cancer cells and they will be lost. And that's a typical phenotype in cancerous, in all of them, the more aneuploid they are, the more necrosis you get. These are dead cells that have actually karyotypically suicided. So this karyotype theory then postulates that clonal, yet flexible karyotypes are the genomes of cancers, not mutations, but genomes mean, I mean now, that part of the chromosome or the genetic material that makes it a cancer cell. It's the karyotype as a whole, just as the karyotype as a whole makes us either a monkey or a dog or a cat or a mosquito or a human. It's not a mutation. You can mutate us as long as you want, you will never get a monkey out of it. You have to rearrange the chromosomes, like you, let's say you are a Volkswagen, you want to make a Porsche instead of an SUV, you cannot mutate the Porsche. You have to rearrange the assembly lines and that's the equivalent of the chromosomes and that's whatever Darwin or God or whoever was at work. Inspiration has done. Here's a graphic of the whole stem. Here's the normal karyotype with diploid stable cells. In step one, aneuploid arises induced either by a casinogen, which I would call an aneuploidogen from a cancer point of view or sometimes spontaneously by an accident. And then you have randomly aneuploid karyotypes which are stable and they keep changing themselves on their two stable end points only. This one is the most stable, that's where most of them end up. They end up in a coffin because sooner or later a chromosome is missing that is essential for the viability of the cell and it dies. And occasionally, as I said, like in roulette, that arrow is thin for that reason, you come up with a cancer cell and that would be a cancer specific karyotype now. A true model indeed for evolution. And once this happens, the cancer karyotypes continues to evolve here. It was originated in an aneuploid pool of karyotypes. Here's the cancer cell and now it has a certain degree of flexibility depending on the karyotype and moves on over many generations. And occasionally, again, it makes an additional evolutionary step when it's challenged by chemotherapy, then a drug-assistant combination comes up that differs from the parental karyotype or a drug-assistant or metastatic one comes up that again differs from the parental karyotype. Although the basic karyotype is maintained, it is adjusted very much like we go from a monkey to a human or a foreword or backwards whatever the direction is. So here's this one experiment that I was going to mention and then a toolie shut up to test the cancer theory, a karyotypic theory. We have asked whether activated oncogenes that have been tested for so long now do indeed induce new tumorogenic cells with individual cancer specific karyotimes much like new species. Moreover, we asked whether the different tumorogenic cell lines arising from human cells consfected with the same oncogenes. I said in these experiments you consfect millions of cells to get a few transformed clones. So it's a very low efficiency. We would predict if it's an evolution, a spontaneous evolution of a karyotype that each of these clonal new cancers that comes out of a homogeneous pool of human cells all consfected with the same oncogenes would each have their own karyotype. Like all progeny of whatever the original mammal was has a karyotype that is in part still conserved in all their derivatives. And again, like individual species evolving from the same ancestors having individual karyotypes with or phenotypes. So the relevance of this for testing the mutation theory is that if individual tumorogenic lines from the same parental cells with the same oncogenes have individual karyotypes and phenotypes, it would follow that the oncogenes have played only an indirect role in transformation as postulated by our theory. It's the karyotype that makes it the cancer cell when the oncogene was essentially like a casinogen. It's like the radiation 30 years before you get the cancer from a Hiroshima or Nagasaki bomb. By contrast, the mutation theory would predict that tumorogenic cell lines show up from these transfected cells with normal karyotypes. They don't spell it out, but they don't say it should change the karyotype. And would have the same phenotypes because the oncogenes they add are encoding these phenotypes. So indeed, we found what the karyotype theory predicts, and that will be shown on my last slide now. We found that different tumorogenic cell lines rising from human cells transfected with the same set of oncogenes have individual clonal karyotypes and phenotypes. That's essentially prognostic cancer experiment here. So here you see two cells or two cell lines that's just under the microscope. You have to look in the animal, you could probably get more different phenotypes, but you can see these cells are phenotypically, morphologically in a pitviddish, quite easy to distinguish. Yet they're generated by exactly the same oncogenes in human epicellular cells, normal epicellular cells. So these phenotypes cannot be so different from the same oncogene, must be something else. And here you can see what it is. It's these two karyotypes that belong to the two cells we see, they're quite different. And they came from this. These were the cells that were transfected. The oncogenes were added and generated aneuploidy, which you could see, a random aneuploidy, didn't show it here. And then you isolate clonal colonies in agar or in animals or in petri dishes. And these are two of these clonal colonies, a very different pattern. And here's a similar example of two different products, again generated with the same set of oncogenes. So that brings me to my last slide. In some of our experiments, confirm the theory that the genomes of cancer cells are flexible clonal karyotypes other than specific mutations. And I thank you again for your attention. Ready for questions. Thank you. Thank you. Thank you. Thank you. Thank you.