Biology 166 Unsolved Problems in Cell Biology

Albert Harris

 

Cancer in One Easy Lesson

"Follow, Poet... follow right... to the bottom... of the night." W.H. Auden

 

1) The disease cancer results from a loss of control of growth and motility, usually resulting from somatic mutations, but sometimes resulting from virus proteins that block normal mechanisms of growth control, for example at cell cycle checkpoints.

2) Please notice that cancer cells do not grow or divide faster than normal cells, although many people believe that, and most forms of chemotherapy were designed on the assumption that they grow faster. Actually, what makes cells cancerous is the lack of control of cell growth, so that they keep on growing without limit, even if slowly. On the other hand, some kinds of cancer cells do grow quite rapidly, even if not faster than certain normal cell types, such as those in the bone marrow that constantly replace blood cells, and those in the intestinal lining and skin (including hair follicles) that constantly renew those structures. In one form of lymphoma, the abnormality has nothing to do with growth or division at all, but instead is a lack of apoptosis (programmed cell death), so that the cells accumulate without limit.

3) Benign versus malignant:When the growth of cells is uncontrolled, but the cells do not migrate or otherwise spread abnormally, and so remain together in a mass, they are said to be benign.Despite the usual connotations of the word benign, a benign tumor is not good for you - and can even be fatal if located in certain places like the brain, where it cannot be removed and where its growth alone can fatally damage indispensable tissues. When cells not only lack growth control but also move without control, in the sense of penetrating other tissues and otherwise spreading through the body, then they are said to be malignant. Lymphomas and leukemias inherently spread in this way, but vary widely in the degree to which they colonize bone marrow, lymph nodes etc.
Tumor versus cancer:The word tumor refers to a mass of (either) benign or malignant cells.
If they are malignant (i.e. invasive, capable of spreading), then the mass is a cancer.
Uncontrolled cell growth: --> benign tumor
Uncontrolled cell growth PLUS uncontrolled cell locomotion: --> malignant cancer

4) Metastasis: (plural, metastases): when cells spread from place to place within the body by invading blood vessels or lymphatics and being carried to new locations by the flow of the blood and lymph, this process is called metastasis. When cells colonize new locations by fluid transport, each of the new masses of cancerous cells is called "a metastasis". Less commonly, cells can also metastasize (that's the verb) by penetrating into other fluids, such as the peritoneal fluid or even the urine in the ureter, and are carried to new locations by these fluids instead of the blood or lymph.

5) Any cell type that is capable of division and locomotion is potentially capable of becoming cancerous. Although cancerous cells tend to be less well differentiated than normal cells, they almost always continue to have many of the differentiated characteristics of the cell type from which they were derived. For example, liver cancer cells still remain recognizable as liver cells (make liver enzymes, are shaped like liver cells, etc.), even after they may have metastasized to some other location in the body.

6) Carcinoma: a cancer derived from epithelial cells and having some of their morphological characteristics. About 80% of cases of human cancers are carcinomas.
The word adenocarcinoma refers to those with a glandular morphology.

7) Sarcoma: a cancer derived from mesenchymal cells, and having some of their morphological characteristics. Fewer than 5% of human cancer are sarcomas; but this percentage is higher in other species.

8) Leukemia: a cancer of one of the many kinds of white blood cells (even including lymphocytes), in which the percentage of these cells in the circulating blood rises greatly. About 28,000 new cases per year occur in this country. The most rapidly growing forms, acute childhood leukemia used to be invariably fatal within a few months, but are now the most curable by chemotherapy. Red Skelton's son and George Bush's daughter, Robin, died of acute childhood leukemia back in the 1950s, but could probably have now been cured with chemotherapy, which has now cured hundreds of thousands of children.

9) Lymphoma: a cancer of lymphocytes.Cancerous cells of this type usually accumulate in the lymph nodes and bone marrow, rather than circulating. There are B-cell lymphomas as well as T-cell lymphomas. The most common form is called Hodgkin's disease, affecting about 7,500 Americans per year, and is about 75% curable by chemotherapy. The other ten kinds are lumped together under the name "non-Hodgkin's Lymphoma" with about 35,000 new cases per year in this country . Many forms are curable by chemotherapy, or go into very long-term remission. Chancellor Hooker died of this.

10) Multiple myeloma is a cancer of relatively well differentiated lymphocytes, in which antibody light chains, or fragments of them are produced in considerable quantities, accumulating in the blood and urine and called "Bence-Jones proteins". The realization that these proteins are antibody fragments, accompanied by the determination of their amino acid sequences, was a crucial step in the history of immunology, and part of what convinced people that antibody specificity results from primary structure and that different antibodies are produced by different clones of lymphocytes. About 12,000 Americans per year die of this disease, of which one of the frequent symptoms is gradual erosion of the skeleton.

11) Teratomas, and teratocarcinomas: are suspected of being cancers of primordial germ cells. These tumors contain many different kinds of differentiated cell types. For example, it is not unusual for them to have brain or kidney cells on the inside, but have hair and/or teeth growing out of their surfaces. There are some special kinds that consist of just neural crest-derived cell types.

12) Neuroblastomas: It so happens that nerve cells never divide, or even synthesize any more DNA, once they have started to sprout axons and dendrites. So there are no cancers of nerve cells. Nevertheless, there is such a thing as a cancer of nerve cell precursors, and such cancers are called neuroblastomas (because a nerve cell precursor, a cell which has become determined to differentiate as a nerve cell, but hasn't actually differentiated yet, is called a neuroblast). There are neuroblastoma cell lines that you can grow in tissue culture; by changing their culture conditions, you can cause the cells to sprout axons, but then they can't grow any more. Sometimes neuroblastomas in children undergo spontaneous remission (self cure) because all the cells of the tumor differentiate to form what amounts to a gigantic abnormal ganglion. One approach to trying to treat this kind of cancer is to induce their differentiation.
There are some even rarer forms of cancer made up of precursors to muscle cells, including skeletal muscle myoblasts, which never make DNA once they fuse and become multinucleate.

13) Mortality: More than a million new cases of cancer per year occur in this country, with around a quarter of these being caused by smoking. In addition, there are around a half-million additional cases of skin cancer; but because these are usually detected earliest, and are also easiest to remove, skin cancers are almost always fully cured, to such a degree that they usually are no longer even included in the cancer statistics. Cigarettes kill slightly over 400,000 Americans per year (almost 10 times as many as by AIDS!), with many of these deaths due to heart disease and emphysema.
There are 150,000 new cases of breast cancer per year; 155,000 cases of colon and rectal cancer etc.
Your chances of getting cancer are about 1/4; your chances of dying of cancer are more than 1/5.
Cancers sometimes kill directly, for example by constricting some vital duct; but more often they kill by weakening the patient so much that fatal infections occur, or heart attacks, or strokes. In the case of weakened patients, the specific diseases involved are often the same ones that kill AIDS patients. Chemotherapy can itself also have fatal results, either directly, by weakening the immune system, or inducing another cancer!

14) Cure: Within living memory, tuberculosis used to be nearly incurable, killed more Americans per year than cancer, and had the same reputation as cancer for inexorable killing. Tuberculosis was what killed Anton Chekhov, Robert Louis Stevenson, the mathematician Riemann, all 4 Brontes, Chopin, Emerson, Kafka, Keats, D.H. Lawrence, Thoreau, Thomas Wolfe and George Orwell, most around age 40. A diagnosis of tuberculosis used to be an inexorable death sentence, but this disease then became almost completely curable with the drugs streptomycin and isoniazid. Unfortunately, Reagan's public health cutbacks (right-wing!) during the 1980s, combined with (left-wing!) soft-headedness toward forcing medical treatment and quarantine of "The Homeless", resulted in production of incurable strains of tuberculosis (because poor patients took too little of the drugs to kill all their germs, but enough to select for mutant germs less susceptible to these drugs). Tuberculosis now kills several thousand Americans per year & well over one million per year world-wide!
Will cancer ever become as curable as tuberculosis did? Maybe you can help make it so. Somewhere, there are weird little facts about cancer cells, that don't seem important because nobody has had the imagination to see how to take advantage of them to kill cancer cells while not harming normal cells. What is lacking is someone with the patience to collect such odd and useless facts, the imagination to figure out how to put these facts to use, the energy to develop methods for using these facts, all combined with the stubbornness not to give up along the way. This person might be you.
Since it is the cancer cell that is abnormal and defective, justice requires that it should die, not you. It is kind of ridiculous that developing the wrong kind of defectiveness in just one of your trillion-plus cells should be fatal to you. We need to find ways to make these kinds of cellular defectiveness fatal for the individual cells that possess them, instead of being fatal for your whole body! Keep this in mind: The goal is find drugs or other treatments that are more poisonous for cancer cells than they are for normal cells!

15)Current evidence indicates that the great majority of human cancers are caused by somatic mutations in certain specific genes. Genes with this property are called "oncogenes", and more than 70 kinds of oncogenes are now known. Based on the increasing frequency with which new methods for finding oncogenes have been finding the same ones over and over again, it is estimated there are upwards of 100 different kinds of oncogenes, total!

16) Somatic mutations, of course, are any changes in the genetic information in somatic cells (i.e. cells other than sperm or egg cell precursors). Somatic mutations are very common (millions or billions of them occur in a normal person); they are passed down only to the daughter cells of the cell undergoing the original mutation, and to their daughter's daughter cells, etc. thus forming a clone of genetically different cells within the body. Nearly all somatic mutations are harmless, either with no effect or making the affected cells slightly abnormal (defective but not dangerous).
But it is very, very dangerous to over-express or otherwise change that tiny minority of genes that code for the proteins whose function is to control cell growth. Cancer can result from single base changes in some of these genes, but is more frequently caused by gene duplications, deletions, or by translocations of genes from their normal location. Translocation to a position adjacent to the control regions (promoters and/or enhancers) of other genes can cause the translocated genes to be 'turned on' (transcribed) when they shouldn't be, or more than they should be. This causes most lymphomas.

17)Almost all cancers are believed to be clonal in the sense that all the cells of a given person's cancer are descended from just one original somatic cell that underwent a mutation in one (or more) oncogenes. The evidence includes such facts as that, in female mammals (where only one of the two X chromosomes is active in any given cell, with half the somatic cells having one of the Xs active and the other having the other X active) all the cells of any given tumor have the same active X. Likewise, cancers in chimeric animals (such as tetraparental mice) are found to contain only cells derived from one of the original embryos from which the animal was formed. In other words, cancer does not spread from cell to cell; the enlargement of tumors does not involve any kind of recruitment of previously normal cells; and it is not "catching".

18) Only certain genes can be mutated in ways which make the cells possessing them cancerous. Mutated genes that have this effect are called "cellular oncogenes", & the normal versions "proto-oncogenes".

19)Paradoxically, oncogenes were originally discovered because of their introduction into cells by certain oncogenic viruses (i.e. viruses that can convert normal cells into cancerous cells). From a historical point of view, it is interesting to note that this outcome was contrary to the natural expectations of most people, such as those in government, who were thinking more along the lines of ordinary viral diseases. Although for some animals (including cats and chickens) communicable viruses really are major causes of cancer (so that anti-cancer vaccines have been developed), such cancers are rare in humans. Many cervical uterine cancers seem to result in part from sexually transmitted viruses; Burkitt's Lymphoma can be caused by the Epstein-Barr virus; "hairy cell leukemia" seems to be caused by a relative of HIV. But the great majority of human cancers seem to be due to somatic mutations. Nevertheless, the discovery of the nature of these mutations depended (in a historical and also a financial sense!) on studies of the molecular mechanisms by which oncogenic viruses cause cells to become cancerous. Without the familiar old simple-minded paradigm ("identify the germ, make a vaccine, etc.") to motivate them, politicians might not have been able to see the value of funding this viral research; and some of them (e.g. Pat Schroeder, John Dingell) have said that the money must have been wasted since the research didn't turn out the way they expected!
Although they are wrong to think the money was wasted, all too little thought has yet been given to the question of how to turn this knowledge into new kinds of treatments: how to design a drug that will selectively kill just those cells containing overactive oncogenes. Probably most cancer researchers think such efforts would be premature; but cancer patients wish they would get on with it. Maybe youwill do it!?

20)Oncogenic viruses have been discovered among at least seven of the dozens of taxonomic groups of viruses, with six of the 7 being DNA viruses. These six produce cancer by causing the over-expression of normal host oncogenes (in one of several ways, to be discussed below in #27). The 7th group were the retroviruses, the RNA viruses that copy their RNA genome into DNA. It turned out that (usually) these cause cancer by carrying copies of human genes that they had picked up from cells their ancestors had previously infected!!! . (Rather than being parts of the normal virus genome (such as gag, pol or env).

21)Oncogene-containing viruses provide a useful means for experimenters to transform large numbers of cells quickly and simultaneously, and with the same biochemical changes in each cell so as to make it practical to study the molecular nature of the changes produced. "Normal" human cancers result from the mutation of one or more proto-oncogenes to over-active forms; but researchers usually can't wait around for some rare mutation to change one cell at a time, especially since each transformed cell would usually differ in respect to which particular gene is over-active. Other methods for discovering oncogenes (besides using viruses) have been developed, including molecular mapping of whatever genes lie next to the reattachment points where chromosomes have broken and become spliced back to the wrong location (as in most lymphomas).

22)The very first virus to be discovered to cause cancer was the "Rous sarcoma virus" (RSV); this was discovered about 1910 by Peyton Rous, although he had to overcome so much skepticism that he did not receive the Nobel Prize for this discovery until 1966, when he was very old! This was also the first of the oncogenic viruses that was discovered to contain an extra gene (in addition to the virus' own genes) responsible for actually causing the transformation.

23)The names given to oncogenes usually consist of 3 letters. For example, the one carried by the Rous sarcoma virus is called by the name src, which is pronounced as if it were "sark". Other important oncogenes include ras, sis, myc and mos. To distinguish between viral genes and their host equivalents, the viral forms are called "viral oncogenes" (e.g. v-src, v-sis etc.) as opposed to "cellular oncogenes" (c-src, c-sis, c-mos etc.). These normal equivalents are also called "proto-oncogenes", but this terminology is more appropriate to the situation where the cancer has been caused by somatic mutation, rather than by duplication or over-expression.

24)"Anti-oncogenes" (also called "tumor suppressor genes") are a special type of oncogene.Do not be misled by the name to believe either that these genes must be the opposite of oncogenes, or that they must in some way counteract oncogenes.The distinction is more like that between dominant and a recessive mutations, with most oncogenes being dominant, and anti-oncogenes being recessive. In other words, it is the under-activity of the anti-oncogenes that makes the cells possessing them cancerous, rather than over-activity having this result, as is the case with the great majority of oncogenes. As one might suspect from this, both copies of such an anti-oncogene gene (i.e. the copies on both of the 2 sets of chromosomes) have to be inactivated in order for the cell to become cancerous.
By far the best-studied anti-oncogene is the one called rb (or rb-1), which causes retinoblastoma in humans. This runs in families and results in eventual blindness due to the formation of multiple tumors in both eyes (in the person's 30s or 40s). Such persons lack a functional rb gene on one of their two sets of chromosomes, so that any retinal cell which undergoes a sufficiently deleterious mutation in the remaining functional copy will give rise to a clone of tumor cells.You might ask yourself what it implies about the underlying mechanisms that such people usually develop multiple tumors in both eyes, but not in other tissues like the skin, or the liver. Another anti-oncogene is called p53, although some mutants of this gene act as if they were ordinary dominant oncogenes, probably because the mutant proteins interact with the normal p53 proteins and prevent their function.

25) More examples of oncogenes:

sis:Codes for a form of PDGF (Platelet Derived Growth Factor). PDGF normally serves as a cell-to-cell signal, secreted by platelets and diffusing to other cells such as fibroblasts and smooth muscle cells, which it stimulates to grow and crawl about. If a cell produces its own form of PDGF, it behaves as if it were being constantly exposed to high concentrations of external PDGF; in other words, it stimulates its own growth and locomotion without limit. This is called autocrine stimulation. It is thought that the sis protein binds to the PDGF receptor proteins while these receptors are in the cytoplasm (prior to their insertion into the plasma membrane where their normal interaction with PDGF would have occurred).

erbB: Codes for an abnormal version of the membrane receptor for the extracellular protein called epidermal growth factor. This form of the receptor behaves as if it were constantly bound to molecules of its growth factor, thus constantly sending a false signal stimulating cell growth. A similar oncogene, called erbB-2 seems (based on its base sequence) to code for a receptor for some other (still undiscovered) growth factor. It is found in amplified form in about one fourth of all human breast and ovarian cancers!

ras:The function of its normal equivalent protein is to relay and amplify stimulatory signals, such as those from growth factor receptors. It binds a molecule of GTP whenever it is itself stimulated. It then relays stimulatory signals and continues to do so until its GTP is hydrolysed to GDP. Certain specific amino acid substitutions eliminate this protein's ability to hydrolyze bound GTP, however, so that it remains permanently in its "on" state, constantly "relaying" non-existent signals for the cell to grow and divide. Such mutations of this one oncogene are believed to be responsible for no fewer than one fifth of all human cancers, including up to half of colon carcinomas, and 90% of cancers of the pancreas! (Although note that several different ras genes are known.) Out of 25 average people, ras will kill one of them!

src: Codes for a protein that spontaneously becomes concentrated on the inside surface of the plasma membrane, especially at the sites of cell-substratum adhesion. This protein is an enzyme (a tyrosine kinase) whose effect is to catalyze the covalent bonding of phosphate groups onto the hydroxyl group of tyrosine amino acid residues of proteins. The proteins phosphorylated by the src protein include some which participate in the mechanical linkage between the actin cytoskeleton and materials to which the outside surface of the plasma membrane attaches; these changes may thus be responsible for weakening the cell's adhesiveness. Work in Patricia Maness' laboratory in the UNC Biochemistry Department indicates that the normal form of the gene functions in the chemotactic guidance of nerve growth cones.
The src protein was the first tyrosine kinase to be discovered. Since all previously discovered kinases (there are many of them in the cell) had catalyzed the bonding of phosphates to the hydroxyl groups of serines and threonines, it was first assumed that phosphorylation of tyrosines was peculiar to cancer cells. But many normal tyrosine kinases have subsequently been found.

myc:Codes for a nuclear protein whose normal function seems to be as some kind of a transcription factor promoting cell growth. For example, when a normal cell is stimulated to grow and divide (for example, by exposure to PDGF), then the c-myc gene product (protein) temporarily increases in concentration; conversely, this gene normally becomes inactive in non-mitotic cell types. Many human cancers have been shown to have undergone amplification of the c-myc gene (often about 10 copies of the gene) this includes many cases of leukemia and about 30% of lung cancers of the highly lethal "small cell" type and breast cancers. The progression of cancerous cells to ever more and more aggressive states is frequently traceable to further amplification of the myc gene. Most cases of Burkitt's Lymphoma seem to result from the accidental splicing of one of the myc genes next to the enhancer (control) sequences of the gene for the heavy chain of M antibodies (on the 14th chromosome). Thus, when cells "try to turn on" the genes for making antibodies, the actual result is to turn on transcription of the growth promoting myc genes. The propensity for the promoters and enhancers of antibody genes to become translocated next to myc and other oncogenes is an unfortunate side-effect of the use of DNA splicing as a mechanism for generating diversity in the genes for the variable sequence regions of antibody molecules. The cutting and rejoining sometimes occurs in just the wrong places. Such translocations are probably the cause of most lymphomas (lymphocyte cancers). Three different versions of the myc oncogene have been described (including N-myc and L-myc); they are believed to correspond to distinct proto-oncogenes, perhaps normally serving different functions related to the control of cell growth. Among their other functions is stimulation of apoptosis.

bcl-2:This name stands for B cell lymphoma and the protein for which this gene codes seems to have the normal function of inhibiting the spontaneous death of B-lymphocytes. In order that the total number of B cells in your body does not continue to increase without limit (because of constant exposure to different antigens) it is essential that the great majority of B-cells self-destruct. This self-destruction phenomenon ("apoptosis") will be discussed more fully below. When too much bcl-2 protein is produced in a given B cell, this blocks the self-destruction. Trans-genic mice with duplications of the bcl-2 gene accumulate abnormal concentrations of lymphocytes, among other abnormalities. Many human lymphomas result from promoter or enhancer sequences of the antibody genes (on the 14th chromosome) accidentally becoming spliced to the site on the 18th chromosome where the bcl-2 gene is located; whenever these B cells "try" to make antibody molecules, what they make instead is lots of the bcl-2 protein. These cells therefore accumulate to form a slow-growing lymphoma (which is always fatal, although not usually until one of the bcl-transformed clones has subsequently also been transformed by an over-activity of the myc oncogene). Prior to this they grow slowly, making it especially paradoxical that these lymphomas can be caused to shrink almost to nothing by the use of growth-poisoning chemotherapeutic drugs.

p53 is sometimes considered an anti-oncogene, in that cancer can result from decreases in its expression. However there are also many cases where cancer is accompanied (and caused ?) by altered forms of this . Science Magazine once suggested that the p53 protein might be the single most important molecule of the decade. It was first discovered as a cancer antigen; because some cancers produced so much of it, and perhaps also because it was present in a modified mutated form, antibodies were made against it. Its normal function is apparently as part of cell cycle control mechanisms for detecting damage to DNA (for example by X-rays) and serving to block the cell cycle at the onset of DNA synthesis until the damage can be repaired. It also has some effect on controlling apoptosis, acting in combination with myc genes.

26) Note that most of the oncogenes listed above are part of a "chain of command" by which external signals, especially protein growth factors, normally stimulate cell growth and division A typical sequence of events would be for a (A) growth factor molecule to diffuse up to a cell's outer surface, then (B) bind to a membrane protein that serves as a specific receptor for that growth factor, with the conformation (C) or other properties of this receptor being changed by the binding, thereby causing either the activation of a cytoplasmic enzyme (that might be a protein kinase or ), or (D) the activation of a g-protein (such as the c-ras protein) that would then (E) activate a protein kinase, which would phosphorylate various cytoplasmic proteins, including some involved in cell adhesion, and would also stimulate increased transcription and translation of (F) genes for certain transcription factors such as c-myc. Paradoxically, myc also tends to stimulate apoptosis (G), unless counteracted by other gene products such as bcl-2.

27) Cancer cells from actual patients usually contain over-active versions of several different oncogenes, not infrequently 3 or 4 or more. Typically, there is one that acts at the nuclear level (such as myc) and one or more (such as ras or src) that act at the cytoplasmic level. For several years (between about 1982-86) it was confidently believed that cancer wouldn't occur unless there were at least two, and that one of the two had to be cytoplasmic in its action (like sis, erb, ras or src) and that the other had to act at the nuclear level (like myc). But this is no longer believed.
Assuming that each oncogenic mutation is itself a rather rare event (3 out of 4 people apparently go through their entire lives without having even one such mutation in any of their trillion+ cells!), you should be wondering how more than one of them can arise with any appreciable frequency in a given cell line. The probable answer is that the initial oncogenic change somehow causes an increase in the frequency of subsequent mutations and (in particular) chromosomal breaks and rearrangements. The loss of growth control promotes all sorts of genetic changes, especially chromosomal breaks and the accumulation of extra copies of chromosomes. Most lymphomas are (eventually) found to have many different chromosomal breaks and translocations, sometimes dozens!

28) Cancer can also be produced by viruses by mechanisms other than the introduction of oncogenes:

    * Strong promoter or enhancer sequences (of the viral genome itself) introduced adjacent to cellular proto-oncogenes; the avian leucosis virus acts in this way.

    ** Inhibitory binding of viral proteins to anti-oncogene proteins; this is what occurs in transformation by SV-40 and polyoma viruses, which are among the oncogenic DNA viruses. (e.g. the rb protein)

    *** Induction of chromosomal rearrangements, causing strong promoter or enhancer sites to become adjacent to proto-oncogenes (as in Burkitt's lymphoma, where the Epstein-Barr virus causes the immunoglobin heavy chain promoter to become spliced immediately upstream of the myc gene)

    Note that the consequence, in each of these cases, is still the over-activity of one of the genes coding for a protein whose normal function is in the same sort of "chain of command" discussed above.

29) Carcinogens: any substance (or treatment, such as x-rays) that is capable of converting normal cells into cancerous cells is said to be carcinogenic, and such a substance is said to be a carcinogen.
It has turned out that almost all carcinogens are also mutagens. i.e. that they cause mutations, including chromosome breaks as well as changes in DNA base sequences. From the descriptions of oncogenes above, this should make sense. There have been some puzzles, however, especially that one major class of some of the most highly carcinogenic compounds, the polycyclic hydrocarbons (methyl cholanthrene, etc.), seemed not to be mutagenic. This was a long-standing puzzle, because this class of compounds were the first carcinogens to be discovered; also because they are the main carcinogens in cigarette smoke, so that the lack of a molecular explanation could be played upon by the paid liars over at the tobacco institute in Raleigh. The solution to the puzzle turned out to be that the body has enzymes whose function is to "detoxify" harmful alien compounds, and some of these enzymes oxidize polycyclic hydrocarbons in a way that converts them to epoxide derivatives, and these derivatives are mutagens. The amounts of these particular detoxification enzymes ("aryl hydroxylases"), turn out to vary genetically from one person to another; and it is believed that your risk of getting cancer from smoke varies in proportion to how much of these enzymes you have.

30) The physical properties of some materials can sometimes make them carcinogenic probably because of their irritant effects on cells that phagocytize them or touch them. For example, particles of asbestos are very powerful inducers of an otherwise rare form of cancer called mesothelioma. No one really understands why, except that it cannot be the chemical properties of this mineral, since it is only effective if the particles are of a certain size range! There are some plastics that reliably induce cancer if small sheets of these materials are surgically implanted into an animal's tissues; but if small holes are drilled through the implanted sheet, then no cancer will be induced! Figure that out, if you can; no one else has been able to.

31) Genetic hybridization can sometimes cause cancer. The best studied case is hybridization between swordtails and platyfish. Backcrosses to the swordtails develop progressively more malignant tumors made of a multinucleated melanocytes. This cell type is normally found in platys but not swordtails.

32) Morphological abnormalities of cancer cells: Cancer cells are usually somewhat abnormal in shape and structure, as seen under the microscope in histological sections. The well known Pap test depends on this fact, although it uses tissue scrapings rather than sections. It is not unusual for cancer operations to begin with the surgeons cutting out a small chunk of the tumor, then sending it down the hall to the histology lab for quick fixation, sectioning, staining and examination by a skilled pathologist. Just by looking at the shapes of the cells and particularly the shapes of the nuclei, the pathologist is supposed to be able to tell whether the tissue is malignant or not. Often there are 3 options: (1) stitch up the incision, because the tissue is benign; (2) continue with the operation and try to remove as much as possible of the tumor, because it is malignant, or; (3) stitch up the incision, because the cells are so extremely malignant that experience has proven that there is no hope from surgery.
The patient, lying there waiting, and his family members waiting down the hall, might be asking themselves what cellular differences the pathologist is looking for. Several large books have been written summarizing the criteria used for this purpose. The quotation below is from "The Cytological Diagnosis of Cancer" by Ruth M. Graham, 3rd. edition Saunders, Philadelphia.
(Health Sciences Library QZ 241 G 741 1972 ) page 379:

    "Whether a cell is malignant or benign is determined by its nucleus; what type of malignant or benign cell it is determined by the cytoplasm."
    "In examining a cell, the microscopist should first look at the nucleus and decide whether it is benign or malignant."
    "The first feature to look for is the orderly arrangement of the chromatin. Are the chromatin particles of equal size? Are they distributed evenly throughout the entire nucleus? Is the nuclear border smooth and even in thickness? Does each part of the nucleus resemble every other part? If, in the mind's eye, the nucleus were bisected, would the two halves be mirror images of one another? If the answers to these questions are "yes", then one can be sure the nucleus is benign. On the other hand, if the answers to these questions are "no"; if the chromatin particles differ in size; if they are distributed unequally at the nuclear border, and in the bisected nucleus no part is a mirror image of any other part, then one can be sure that the nucleus is malignant. " (emphasis added)

Note that these criteria are entirely empirical (arrived at purely by experience, not based on any theory or other reason for expecting them). Cells with one set of properties always turned out to be malignant in their future behavior (if not removed), while cells with the other sets of properties always turned out to be benign. Mistakes are sometimes made, even mistakes in the direction of diagnosing malignant cells as benign; but given the amounts of money involved in malpractice lawsuits, it seems noteworthy to me that the quotes above could be so general and sweeping. It is almost as if the weather could be accurately predicted from the shapes of clouds, but no one had bothered to find out the physical causation relating the shapes of today's clouds to the occurrence of tomorrow's storms! There has been little or no research into the question of how cell and nuclear shape is related to oncogene function. In fact, the trend among cancer surgeons is to get away from using these morphological criteria, mostly in favor of fluorescent staining with monoclonal antibodies. Duke recently lost a mammoth set of malpractice lawsuits resulting from misdiagnosis of benign tumors as malignant, based on staining with a certain monoclonal antibody. The histological organization of cancer cells is also abnormal; one might describe the cells' arrangements as "sloppy": they are irregular in shape, sizes and relative positions, as well as slightly out of alignment. As compared with the normal tissues, sections of malignant tissues look as if they had been poorly drawn by either an incompetent or a lazy artist. What does this imply about the mechanisms that control cell shape, size, relative position, alignment, etc.? Would you prefer to say that the cancerous state interferes with these mechanisms? Or would you say that malignancy results from the disruption or failure of these mechanisms? Cancer cells also tend to show a reduced degree of differentiation, although it is no longer thought correct to think of them as having reverted to the embryonic state, much less as having remained in that state (which were once popular theories).

33) Cancer cells would not be malignant unless their locomotion were also out of control, not just their growth. The phenomena of contact inhibition (of cell locomotion) and contact paralysis (of cell surface movements) are believed to be reflections of the mechanism by which the motility of normal cells is controlled, with this control mechanism being part of what is defective about malignant cells. The evidence for this latter conclusion was that several kinds of cancerous cells were shown to be less susceptible to contact inhibition than equivalent normal cells: they overlapped more, they continued forward spreading and ruffling along parts of their margins that had come in contact with other cells, and so on. Such behavior is also seen in normal macrophages and polymorphonuclear leucocytes, which makes sense because it is part of the normal differentiated phenotype of those cell types to penetrate invasively through tissues.
On the other hand, some cell lines that are malignant in vivo seem not to lack either contact inhibition or contact paralysis in culture. Furthermore, it has been argued that contact inhibition depends too much on the nature of the culture substratum, glass or plastic, to be directly relevant to what goes on inside the body. In my opinion, the real obstacle has been the lack of certainty about what actually gets inhibited, at a mechanistic or molecular level, when ruffling is paralysed by contact and a cell stops, turns, or reverses direction, apparently as a result of its "propulsive motor" having been locally inhibited. As was discussed earlier in this course, the evidence now points to actin assembly, and/or the coupling of newly assembled actin to transmembrane "tine proteins" or protein complexes, along the advancing cell margin. The point is that if the "ruffling" that is being paralyzed corresponds to actin assembly, etc., this would imply that contact paralysis amounts to an inhibition of actin assembly near cell-cell adhesions; conversely, failure of this paralysis in malignant cells would imply that part of the invasiveness of cancer cells may result from an increased ability to continue actin assembly near contacted cell margins. This subject obviously deserves more research.

34) Behavioral and structural abnormalities of cancer cells in tissue culture:

    *Reduction or loss of cytoplasmic "stress fibers" (containing actin, type II myosin, tropomyosin etc.)
    *Redistribution of cytoplasmic actin from stress fibers to lamellipodia.
    *Increased numbers of areas of marginal ruffling.
    *Lack anchorage dependence (i.e. can grow and synthesize DNA, even without a solid substratum).
    *Decreased adhesiveness, largely because of reduced fibronectin (the cell-collagen linking protein).
    *Weakened cellular contractility and traction.
    *Ability to spread on less adhesive substrata (presumably because of weakened contractility).
    *Abnormally anaerobic metabolism; secretion of much lactic acid (even with of plenty of oxygen).
    *Continue to grow at serum concentrations much lower than required by normal cells.
    *Immortal in tissue culture; can grow indefinitely in culture without undergoing senescence.
    * ...and many other differences

35) Among the differences between benign and malignant tumors is that the former become surrounded by a "capsule", consisting of a fairly dense wrapping or coating of type I collagen fibers applied to it by fibroblasts in the surrounding tissue. Benign tumors are encapsulated and malignant cancers are not encapsulated.
Most internal organs are encapsulated (e.g. the kidneys, the liver, etc. all have capsules; the outer white layer of the eye is its capsule; the capsule of bones are called "periostea", that of the testis is called the "tunica albuginea", etc.; the dura mater is essentially a capsule; and I can't think of any exceptions right now, in the sense of organs that aren't encapsulated; maybe the ovary?). As a rule, foreign objects embedded inside the body also become encapsulated. For example, I own a dog who has encapsulated some buckshot (administered by someone before we got him). No one knows, and almost no one cares, what the mechanism of capsule formation is, neither in the sense of what physical processes compress the collagen right up against the surface of the organ, bullet. or tumor, nor in the sense of what signals trigger this response by nearby fibroblasts, nor even in the sense of whether the mechanism of capsule formation is the same for normal organs as it is for tumors or for bullets.
The capsule around a benign tumor helps with the removal process: surgeons sometimes speak (rather graphically!) of "shelling a tumor right out". What can one gather from this about the degree of adhesion between tumor and capsule? The capsule also tends to block any incipient invasiveness on the part of the neoplastic cells. I think it is a good question what cellular properties induce nearby cells to form a capsule, and whether the failure of malignant cells to induce such as response is positive or negative (do the cancer cells lack some property that would have called forth capsule formation; or do they have some property that actually inhibits the attempt to make a capsule; or do they have some property that makes it difficult or impossible for the capsule to be formed). Another good question is whether one could artificially stimulate capsule formation by cells near malignant tumors, thereby reducing invasion and facilitating surgical removal later on?

36) Secretion of proteolytic enzymes: Malignant cells typically secrete proteolytic enzymes, or activators of proteolytic enzymes (plasminogen activator). This is believed to be an important part of their capacity to invade adjacent tissues, and (in the case of carcinomas) their ability to disrupt and penetrate their own basement membranes (=basement lamellae). There has been a huge amount of funding, both from drug companies and the NIH, directed toward finding inhibitors of these enzymes; the idea being that such inhibitors might prevent invasion and metastasis.

37) Cancer chemotherapy and the problem of selectivity : The progress of cancer can often be slowed, and its fatal consequences delayed for months or years, and in tens of thousands of people per year, cured altogether by treating the patient with one or more of these poisonous chemicals. This approach is analogous to treating bacterial diseases with antibiotics (which are selective poisons), except that the problem of selectivity is much more difficult. For example, penicillin blocks the bacterial enzymes whose function is to synthesize cell walls, so the bacteria cannot make new cell wall as they grow, and therefore burst osmotically. Animal cells do not have (or need) cell walls, nor do they even have an enzyme comparable to the one inactivated by the penicillin molecule; thus, even high concentrations of this drug do not poison animal cells much. Another example are the streptomycins, which selectively poison bacterial ribosomes, while animal cell ribosomes are sufficiently different in structure not to be harmed. And chocolate is poisonous to dogs but not to people.
There is an excellent book titled "Selective Toxicity" (by Adrien Albert: now out of print!) which catalogs thousands of examples and specific mechanisms where it is possible to poison one kind of organism without harming another.For example, there is a kind of rat poison which blocks blood clotting in rats, but not in humans, and so on. In general, however, the more closely related a pathogen is to humans, the harder it is to kill by chemotherapy; this is why fungal diseases are usually harder to cure than bacterial ones, while protozoan diseases are harder still. With cancer, the problem is even worse because you are trying to kill certain ones of your own cells, and to do so with enough selectivity that you don't kill too many of your normal cells!It is sort of like to trying to kill burglars in your house by poisoning the air or water - how do you keep from killing yourself ? Nearly all the drugs used for cancer chemotherapy poison some aspect of cell growth or division, and are most damaging to faster growing cells. In fact, the paradigm of selectively poisoning cell growth has come to dominate the search for new anti-cancer drugs, to the extent that substances tend not even to be tested for anti-cancer effects unless there is some prior reason to believe that they will poison cell growth.

38) Cancer chemotherapy: Types of actual drugs now used for chemotherapy:

    Nitrogen mustards: (related to the sulfur mustard gasses used in the first world war, and more recently by Iraq): these bond covalently to guanine bases in the DNA and thereby eventually break the DNA chains.

    A commonly used nitrogen mustard is cyclophosphamide ("Cytoxan").
    It is a chemically inactive form, which is taken orally and then converted into an active form by liver enzymes. A few days treatment can totally cure many cases of Burkitt's Lymphoma.
    This drug is also used to treat certain autoimmune diseases, such as multiple sclerosis.
    Other nitrogen mustards include chlorambucil, bisulphan and malphalan They are taken intravenously.

    Nucleic base analogs:

      5-fluorouracil Inhibits DNA synthesis (by inhibiting thymidine synthesis)
      6-mercaptopurine Inhibits adenylate synthesis
      6-thioguanine Inhibits DNA synthesis (because it is an analog of guanine)
      Cytosine arabinoside Inhibits DNA synthesis (because it is an analog of cytosine riboside)

    Other inhibitors of DNA synthesis:
      Methotrexate: An analog of folic acid, thus inhibiting synthesis of nucleotides in general.

    Antibiotics: (synthesized by bacteria related to those that make streptomycin, to which they are analogous)
      Daunorubicin: Binds to DNA
      Mithramycin: Binds to DNA
      Bleomycin: Binds to DNA
      Mitomycin: Binds to DNA, causing fragmentation of chromosomes

    Anti-microtubule poisons: ("spindle poisons")
    Vinblastine: inhibits the polymerization of microtubules by causing tubulin to aggregate into an alternate crystalline state, thereby preventing mitosis.
    Vincristine: also inhibits the polymerization of microtubules.
    Taxol: promotes polymerization of microtubules, blocking their depolymerization (the reverse of preceding 2) (this is the one, much in the news, extracted from the bark of Pacific Yew trees)

39) Side effects of chemotherapy:
    Anemia, due to poisoning of rapidly growing hemopoetic cells of the bone marrow;
    Nausea, due to poisoning of rapidly growing cells lining the digestive tract;
    Hair loss, due to poisoning of rapidly growing skin cells of hair follicles
These three side effects are produced to varying degrees by all of the drugs listed above. Other drugs are given to counteract the nausea, and patients' blood counts and clotting times are carefully monitored.
In addition, certain chemotherapeutic drugs have additional side effects peculiar to them (the reasons for which tend to be much less well understood) For example, some of those listed above under "antibiotics" cause selective destruction of heart muscle cells.
Unfortunately, most of these drugs are also mutagenic, and therefore carcinogenic. Several percent of patients undergoing chemotherapy eventually develop other forms of cancer as a result.
On the brighter side, chemotherapy patients report great improvements in hay fever and other allergies!

40) Relative effectiveness of chemotherapy against different forms of cancer.
In the case of cancer, you obviously wants to find poisons that will kill only the cancer cells, while damaging normal cells as little as possible. This means that there are two aspects to the problem: first, to find out which properties of cancer cells which differ (substantially) from those of normal cells; and second, to synthesize chemicals (or find them in nature) that are selectively toxic to just those cells having the abnormal cancerous properties.
As a rule, the fastest growing forms of cancer are the only ones that can actually be cured by chemotherapy Just as we saw with the 3 side effects listed above, the faster the cells grow and divide, the more likely they are to be killed by the chemotherapeutic drugs. Does this mean that there is no hope for curing the majority of kinds of cancer, which happen to be slower growing?

41) The paradox, a possible explanation, and its implications for improving chemotherapy.
Cancer cells do not really grow faster than normal cells; what makes them cancerous is that their growth is uncontrolled, continuing without normal limits, not that it is faster. Many kinds of cancer cells don't even grow faster than their normal equivalents, and none grow faster than the fastest growing normal cells (i.e. those of the marrow, mucosa and skin). An ideal anti-cancer drug would thus be one that poisoned just those cells whose growth is out of control, regardless of whether this out of control growth was particularly fast or not. We should probably be surprised that the available drugs work even as well as they do. If cancer cells don't really grow faster than normal cells, then how can it be that they cure tens of thousands of people, and lengthen the lives of hundreds of thousands more. Faced with their proven effectiveness, one can either say "don't look a gift horse in the mouth", or one can try to figure out what the source of the extra selectivity might be. After all, if we don't really know why these drugs have as much specificity for cancer cells as they do, then how can we expect to be able to increase this specificity enough to cure the slower growing cancers?

42) "Apoptosis" is the recently invented (1982) word for programmed cell death. Most if not all of the cells of the body turn out to have a built-in self-destruct system, analogous to the explosives in unmanned NASA rockets. A familiar example is the self-digestion of the cells of a tadpole's tail, which is as normal and necessary a part of its metamorphosis as the growth of the legs. A less familiar example (that is a common event in many animals, including humans) is the self-destruction of virally infected cells; this is induced by T-lymphocytes, among whose effects is to activate the infected cells' own self-destruct mechanism. Obviously, if you could find some sufficiently selective way to activate this self-destruct mechanism in cancer cells (to a greater degree than in normal cells), then this could be the basis of revolutionary improvements in cancer therapy. In fact, many researchers now conclude that this is actually how some (or maybe even all?!) of the currently-used anticancer drugs actually work, even though these drugs were developed and assumed to act by direct killing.
In the case of those anti-cancer drugs that now appear to act by inducing apoptosis, the basis of the killing and of the selectivity for cancerous cells seems to lie in a (perhaps somewhat surprising?) mechanistic overlap between the systems for regulating the cell cycle and those for initiating apoptosis of damaged or excess cells. Humans probably wouldn't build-in so much overlap between parallel mechanisms; but Darwinian evolution progresses by using existing genes for new functions. Imagine a nifty fail-safe device designed to remove cars from the highway when their brakes become too defective. A button under the brake pedal sends voltage to a spark plug located in the gasoline tank! As the brakes become weaker, then the driver will push down harder on the brake pedal, eventually pushing hard enough to reach the button that blows up the gas tank. Absurd as that would be in car design, the cell cycle checkpoints (the G1->S one, anyway) seem to contain elements logically equivalent to this, with the equivalent of brake failure being cancer, and at least some anti-cancer drugs acting to "push the button". Although it would make absolutely no sense in terms of the traditional theories of anti-cancer chemotherapy (for which selectivity was thought to derive from faster cell cycling), it is found that lymphomas caused by deficient apoptosis (due to excess bcl-2) are nevertheless very responsive to nitrogen mustards. Why cross-linking guanines in DNA should promote these cancerous cells' apoptosis, and do so more than in normal cells (including those with normally lower levels of bcl-2 gene activity), is one of the great unasked questions of medicine. Note that myc genes also stimulate apoptosis, as well as DNA synthesis. This is more like having the self-destruct button under the accelerator pedal!

43) Studies of the mitotic cell cycle have revealed the existence of several "checkpoints" at which the cell "checks" to see whether the current stage of the cell cycle has been completely finished, and is not supposed to allow the next stage of the cycle to begin unless and until the current preceding stage is entirely completed. For example, one set of checkpoint mechanisms will not allow a mitotic cell to progress from metaphase to anaphase (in other words, will not permit the sister chromosome pairs to separate and move to the poles) until all the chromosomes are aligned between the poles. Cells which cannot form spindles because they have been treated with microtubule poisons will go into metaphase, but just sit there for hours with their chromosomes condensed in pairs. In some studies, it was found that just holding one of the chromosome pairs away from the metaphase plate (with a microneedle) was sufficient to prevent the whole spindle from progressing into anaphase. In fact, it was sufficient to prevent the phosphorylation of certain centromere proteins, which seems to be a necessary part of controlling the onset of anaphase. Another checkpoint mechanism blocks cells from beginning the S phase (DNA synthesis) if some of their DNA has been crosslinked or otherwise damaged. This damage stimulates synthesis of the p53 protein. A third checkpoint mechanism blocks cells from going into mitosis until S period has been completed, in other words until all the DNA of all the chromosomes has been completely duplicated. After all, if a cell divides when some of its genes haven't duplicated, then it will lose those genes from at least half the daughter cells, which is likely to kill those cells if the genes are ones that code for important proteins. And if several such genes, on several different chromosomes, had not been duplicated by the time of division, then both daughter cells would die! Probably what actually kills cells during cancer chemotherapy is failure of checkpoints: not the damage to DNA per se, nor the prevention of mitosis, but rather the continuation of mitosis by cells with damaged DNA, or with defective mitotic spindles, etc. The cells that die are not the ones that are prevented from dividing, but rather those that go ahead and divide anyway, despite not really being ready If this were true, it would mean that the (all too inadequate) specificity of chemotherapy, its ability to kill cancer cells somewhat more than normal cells, is not really a result of differences in growth rates per se, but instead due to defective checkpoints in the cancer cells.

It makes a lot of sense for cancer cells to have defective checkpoint mechanisms; after all, what makes them cancerous is some kind of failure of their growth control mechanisms. Indeed, the ideal treatment mechanism would be one that killed just those cells in which these mechanisms were defective, whether or not they grew and divided rapidly, and not cells that grow rapidly for other reasons. On the other hand, one would have expected that the control mechanisms whose failure causes cancer would not be the ones that monitor readiness for the next stage of the cell cycle, but rather the ones that monitored cell crowding, or organ size, or cell-cell contact, or something like that - thereby blocking growth among crowded cells. I for one would have expected these kinds of growth control mechanisms to be quite different from the ones that monitor such things as completion of DNA synthesis, alignment of mitotic chromosomes, etc. But perhaps there is enough overlap between these two classes of mechanisms so that when one is defective, the other also tends to fail. For example, they might both use some of the same protein subunits, such as G-proteins or kinases or something. It is even possible that both the growth control mechanisms and the cell cycle checkpoint mechanisms "feed" into some common systems for stopping further growth. Evidence for such connections could easily have been missed, however, simply because so much of the research progress concerning checkpoints has depended on studies using yeast as the experimental organism, with yeast presumably having no equivalent to the inhibition of growth by crowding that occurs in the tissues of animals. A high priority should be given to the search for such connections in future research on cancer cells and the reasons for their special susceptibility to being killed by drugs that poison some stage of the cell cycle.

44) Spontaneous remissions & Coley's toxins: It is not as rare as you might think for very serious cases of cancer to undergo spontaneous remission: that is, "just to go away". It happens all too rarely, of course; but it really does happen; and if someone could figure out what causes it to happen, then might become the basis of an entirely new approach to treatment.
In 1892, a young cancer surgeon named William Coley noticed that several of his patients underwent spontaneous and apparently complete remissions from cases of cancer that had been expected to be hopeless. Each of these remissions followed severe bacterial infections, in which the patients had run high fevers. Coley had the idea that he might be able to cure other patients by deliberately infecting them with the same sorts of bacteria that had caused these fevers, or by inducing fevers by injecting patients with extracts of the bacteria. Using this latter approach, he was apparently able to cure hundreds of terminal cancer patients. His fever-inducing bacterial extracts were called Coley's toxins, and were widely used by him and others up to his retirement around 1930. They were also sold commercially for many years by one of the major pharmaceutical companies. Even critics agree that this method cured many patients, probably approaching a total of one thousand people! A combination of factors caused the approach to be abandoned in the 1930s. One problem was the lack of a theoretical basis no one had much idea why the method worked, which meant that they had no idea how to improve it. Another was its lack of patentability; there was no economic motive to continue making the toxins. Some batches worked much better than others, and no one knew why. This treatment is no longer used anywhere.
Reinvestigations of the bacterial toxins at the Memorial Sloan Kettering Cancer Research Center in New York did lead to the discovery of a protein called "tumor necrosis factor". This is one of many cytokines; it is a small protein that (in this case) macrophage cells synthesize and secrete in response to stimulation by (among other stimuli) certain molecules released by the bacteria. TNF, as it is called, stimulated massive breakdown in certain kinds of cancers, in large part by stimulating disorganization of the blood vessels leading to them. TNF has been totally sequenced, cloned into bacteria, etc. and is therefore available in reasonable quantity. Incidentally, its effects are NOT species specific: e.g. TNF can cause breakdown of tumors in mice, and vice versa. But there are lots of puzzles remaining, and it is not yet used in treatment.

45) The interferon's are another class of cytokines (i.e. hormone-like peptides or proteins that are made and secreted by one of the kinds of white blood cells, and serve to stimulated some kind of activity by other cells). There are several different kinds of interferon's (alpha-interferon, beta-interferon, gamma interferon), each of which is made by different cell types and has a different amino acid sequence. All of them stimulate cells to become more resistant to viral infection (they were given their name because they interfere with virus growth). Among other effects, they stimulate cells to have larger amounts of histocompatibility antigens on their surfaces, and also increase the stringency of gene transcription and other molecular processes. Essentially, cells become much more "picky" about which genes they transcribe and which messengers they will translate. When you have "the flu" or other viral disease, part of the way you fight off the virus is by producing lots of interferon. On the other hand, much of the side effects of the flu, including fevers and severe aches, are side effects of the interferon. Incidentally, in contrast to TNF, interferons are highly species specific; the amino acid sequences differ from one kind of animal to another, and (for example) mouse interferon has little or no effect on human cells, or vice versa.
Several of the interferons are now being used in trial clinical studies of human cancer patients. Following completion of chemotherapy, a randomly chosen half of the patients are injected daily with interferon for 1 to 2 years(!), during which time these patients continue to suffer flu symptoms! One of these programs is in the Duke Hematology Dept. Although there have been very promising improvements in cases of some kinds of leukemia, (hairy cell leukemia) these are rare kinds that are suspected of being caused by viruses anyway.

46) Tumor angiogenesis factor, and the hope of preventing the vascularization of solid tumors. Another surgeon, Judah Folkman, noticed many years or so ago that in a few cancers only the outermost surface layer of cancer cells remained alive. The inside cells all died for lack of oxygen and nutrients, because blood vessels had, for some reason, failed to extend into these cancers and vascularize them. This is in contrast to most cancers, which do become vascularized. Folkman developed the idea that it might be possible to treat cancer by blocking vascularization, if only we understood the mechanisms controlling this process. He hypothesized that most tissues secrete a factor that attracts and stimulates endothelial cells to sprout from nearby capillaries; he named this "Tumor angiogenesis factor" (TAF) and has been trying to isolate it and determine its chemical nature. He developed a bioassay in which bits of material are inserted into the clear part of a rabbit's cornea, a part of the body which is unusual in that it normally is entirely unvascularized, but into which blood vessels can sometimes be attracted. His group, which has included many students, has made many discoveries about the vascularization process, and have recently reported curing cancers in mice with this approach, causing huge increases in the company developing this approach.

47) Immunotherapy, vaccines, etc.: As you should know, what eventually cures us of most infectious diseases is the operation of our immune system. And much of modern medicine is based on manipulating the operations of this system, helping it along with antisera and vaccines. So why won't these same approaches also work with cancer? Or can they?
The short answer is "No, they can't.", because cancer cells are part of yourself, and the immune system contains mechanisms to prevent itself from making anti-self antigens. The long answer is that there may be ways to use immunity anyway. In fact, that may have been part of the mechanism of Coley's toxins. The vertebrate immune system uses the splicing of DNA to achieve random generation of antibodies against all possible antigens, followed by a process of 'weeding out' all lymphocytes whose antibodies would attack normal molecules of your own body. The result is called "immune tolerance". Sometimes this weeding-out process fails, and the result is called an auto-immune disease, in which your immune system attacks some part of your own body, often eventually killing you. An example is multiple sclerosis, in which the immune system attacks a certain membrane protein found in Schwann cells, thereby destroying myelin sheaths and nerve fibers and causing paralysis, blindness and so on. So the problem is not that your immune system cannot attack parts of your own body; the problem is that you don't want it to, unless the cancer cells contained some proteins or other antigens not found in any other cells of the body. Actually, this may sometimes occur; some cancers become subjected to fairly massive attacks from the immune system, as if they did have special antigens. But from what you have learned about oncogenes above, it should be clear that this is somewhat surprising: why should over-activity of these normal genes involve production of any totally abnormal proteins or other antigens? Conversely, can you think of any way to cause the loss of growth control also to result in the production of alien antigens of some kind, to which the body is not tolerant. Answers to questions like these could lead to real breakthroughs in cancer treatment. And without answers to such questions, we can't expect too much help from the immune system. To produce a vaccine against a germ, it is usually just a matter of isolating some of the germs' antigens, and injecting them before hand to stimulate the body to get ready to attack molecules of this shape. This won't work if the antigens we want to attack are normal parts of the body; firstly, because we are probably already tolerant to these antigens; and secondly, because if you could stimulate immunity against them, this would kill all the normal cells, too. As with chemotherapy, the problem is specificity.

48) Immunity against bacteria is mostly due to antibodies; but for viruses, most of the work is done by one of the several kinds of T-lymphocytes, which produce special antibody-like proteins on their cell surfaces. These are called "T-Cell receptors", and they are the logical equivalent of antibodies in that they have the same degree of binding specificity, each to its specific antigen; this specificity is produced by a process of random DNA splicing, and so on. But these cells actually attack and kill other cells, being stimulated to do so whenever they encounter another cells that has the appropriate antigen on its surface. Almost always, these other antigens are held out from these other cells' surfaces in the grip of a certain kind of protein called histocompatibility antigens. I am most concerned with the "type I histocompatibility antigens" here. These are normal parts of the outside membrane surface of almost all your cells (except red blood cells). Their function is to hold out fragments of proteins that have been produced by protein breakdown inside the cell. In a virus infected cell some of these fragments held by these holders will be virus proteins. When they are, it is the logical equivalent to holding up a little flag or a sign reading "Please kill me; I am infected with a virus; Will a T-lymphocyte please come and lyse me!". When touched by a lymphocyte whose t-cell receptor proteins happen to have just the correct shape to bind to the combination of holder and antigen, the infected cell is killed. For many (or most?) virus diseases, much of your recovery is due to t-lymphocytes going around and killing infected epithelial cells, etc. (in response to them bravely holding up the little flags!), which you then regenerate.

49) The future: Despite the huge numbers of chemicals that have been and will be tested every year for anti-cancer effects, despite the dedication and tireless efforts of researchers and clinicians, do not assume that "They" will automatically find a cure. It may be that "They" will need your help, because unfortunately "They" are collectively sometimes rather dim, unimaginative, busy-busy, and generally in-a-rut. Coley's toxins were simply dropped. Pharmaceutical companies have responsibilities to their stockholders and are not wasting time looking for types of cures that could not be patented if found; you should not expect them to do otherwise; and don't kid yourself that you would in their place. Furthermore, nearly all bioassay "screens" for anti-cancer capacity depend on blocking growth of fast-growing cells; unless a chemical acts in this way, it will be discarded. Is there some way to promote apoptosis of cancer cells? No one knows; and no one knows how to find out, or has a bioassay for screening substances to find out whether they can induce apoptosis. Interferons are being given to hundreds of human cancer patients, but this has less to do with any rationale for their effectiveness against cancer than with their new availability from biotechnology companies. Meanwhile, government funding for cancer research has been cut way back to help pay for studies of a Certain Venereal Disease. Of the grant proposals that pass screening and are approved for funding, there is currently only enough money to pay for the 20% to 25% with the highest priority. No such grant proposal has any chance of being funded unless it exactly fits current theories as to the molecular nature of cancer. Thousands study oncogenes and their modes of action; but almost no one is looking for ways to kill cells on the basis of the over-activity of oncogenes! At the same time, be wary of quack remedies. Huge amounts of money and hope were squandered by advocates of "Krebiozin", "Laetrile" and other fraudulent remedies. It is no solution to give up careful, critical thinking; but it needs to be combined with imagination and open-mindedness. Where are the William Coleys of today and tomorrow? Could one of them be you?

50) Any consistent difference in any cellular property could potentially be turned into a treatment. All you have to do is impose chemical and/or physical conditions that tend to kill cells selectively, depending on whether the cells possess the abnormalities characteristic of cancer cells. Any consistent difference in any cellular property could potentially be turned into a treatment method. All you have to do is impose chemical and/or physical conditions that tend to kill cells selectively, depending on whether the cells possess the abnormalities characteristic of cancer cells. Any consistent difference in any cellular property could potentially be turned into a treatment method. All you have to do is impose chemical and/or physical conditions that tend to kill cells selectively, depending on whether the cells possess the abnormalities characteristic of cancer cells. Say these two sentences over and over and over. Don't forget.

Cancer: A concise framework for class discussion (please ask what don't you agree with?)

A) In contrast to infectious diseases, where the body is invaded by some other species of organism (bacteria, fungi, protozoa or viruses), cancer consists of "invasion" by mutated forms of the body's own cells.

B) A general cure for cancer would require a drug or other treatment capable of selectively killing just those cells having cancerous properties, and not killing too many normal cells.

C) Cancer results from insufficient control of growth and movement, not from abnormal mechanisms of division or locomotion.

D) Cancer cells do not grow faster than normal cells; no cancer cells grow faster than the fastest growing normal cells (stem cells of skin, gut and bone marrow, or cells in healing wounds). In some kinds of lymphoma, the defect is not growth at all, but not enough programmed cell death!

E) Only a small fraction of our genes can be mutated in ways that will make cells cancerous.

F) All these oncogenes have important normal functions related to the control of cell growth and/or movement and/or programmed cell death.

G) No one has yet thought of ways to kill cells selectively on the basis of the over-activity of oncogenes, or the underactivity of anti-oncogenes.

H) Nearly all existing chemotherapy is based on poisoning cell growth (anti-DNA mustard gas-related compounds, anti-microtubule poisons that block mitosis, DNA base analogs, etc.)

I) Existing chemotherapy therefore has strong side effects on normal stem cells, causing nausea, anemia, hair loss, etc. as well as being ineffective on most slower growing cancers.

J) Searches for new cancer drugs concentrate almost entirely only on chemicals that poison cell growth

K) The unnoticed paradox is that these growth poisoning drugs can cure many cancers, and even kill 99.99% of lymphoma cells whose defect is insufficient programmed cell death.

L) A possible partial explanation for this paradox would be that normal cells protect themselves from being killed by these drugs by slowing-down their rates of growth while the poisons are present, using their normal growth control mechanisms to do this.

M) Based on what you know about apoptosis, do you have any new ideas about how it might be possible to stimulate apoptosis specifically in cancer cells?

N) In regard to the normal growth control mechanisms (such as those that keep organs from growing to large; and/or that stop regeneration once some missing tissue has been replaced); would you expect that these mechanisms would be defective in cancer cells?

O) Furthermore, in relation to these same normal growth control mechanisms in addition to being activated by contact or crowding of cells, might you also expect for these same mechanisms to inhibit cell growth in response to the presence of poisons that damage DNA, block copying of DNA, cause synthesis of abnormal DNA, interfere with formation mitotic spindles, etc.? Why or why not?

P) Unless your answer to both of the last two questions was "Yes", then how can you expect to kill cancer cells selectively using drugs that damage DNA?

Q) And if your answers were yes, than how might the effectiveness/selectivity of these drugs be improved for treating cancer?

R) Could it possibly help the effectiveness of existing chemotherapeutic drugs to combine them with some chemicals or other treatments that stimulate increased cell growth? (including increased growth of cancer cells)

S) Explain why you might expect "frame-shift mutagens" not to be strongly carcinogenic?

T) Can you invent any new approaches to killing cancer cells?

U) Might it help to weaken the mechanism of immune tolerance (whatever that mechanism is)?

V) Might there be some way to cause cancer cells (but only cancer cells) to present non-self antigen peptides in their type I histocompatibility antigens? (Even though they have no specific gene for these peptides)

W) If there are consistent differences in the shapes of nuclei of cancer cells, must this be caused by some abnormal mechanical properties of their cytoskeleton and or membranes?

X) Can you imagine or invent some way to kill cells selectively depending on whether they are abnormal in these properties that cause shape abnormalities in nuclei (that are used in diagnosis)?

Y) Suggest strengths or weaknesses of Folkman's concept of preventing blood vessels from vascularizing tumors.

Z) Can you invent some entirely new way to kill cancer cells without hurting normal cells?

Questions that I don't think people ask often enough:

I) Why does chemotherapy work even as well as it (sometimes) does?

II) Why should it kill a cancer cell to prevent if from dividing? And in the specific case of the supposedly "tailor-made" inhibitors of "rogue enzymes" tyrosine kinase fusion proteins coded for by the "Philadelphia chromosome", what sense does it make for cancer cells to be killed by blocking catalysis by an enzyme that is abnormal anyway? Another more specific question in that particular case is how a pyrimidine could be such a specific inhibitor of a specific ATPase, given that adenine is a purine!

III) Why do most people so willingly accept such assumptions that fast-growing cells can be selectively killed by drugs that block growth? Very often (although not always) fast-growing cells are killed by such drugs; but why shouldn't we expect that such drugs would simply stop them from growing as much, rather than causing them to die? And why aren't we surprised by this?

IV) Specifically in the case of microtubule inhibitors (vinblastine, vincrisitine, taxol, colchicine, nocodazole, griseofulvin, etc.) why are some of these reasonably effective anti-cancer drugs (the first 3 on this list), while others are found to have no anti-cancer effect at all? A simple-minded explanation might be that this difference results from some of the drugs getting into the cells, and cells being more impermeable to the others. But I don't think the explanation is anything like that: as far as I know, for example, mitosis is inhibited by colchicine just as much in cancer cells as in non-cancerous ones. The more important fact is that neither the normal nor the cancer cells die as a result of their mitoses being blocked by colchicine. Why should blocking division cause a cell to die, after all? Perhaps there are some special ways of halting division such that the cell dies as a result, or such that only cancerous cells die as a result, as opposed to other ways of halting mitosis (in non-cancerous cells) such that they just wait until the drug is gone, and then continue with their division.

V) How do the cell cycle checkpoints detect and regulate cell size (=volume per amount of DNA in the nucleus)? Is something (a chemical?) produced that spreads evenly out through the cytoplasm, such that volume of cytoplasm is measurable by the dilution of this chemical? Do growing cells wait (at G1->S?) until the cytoplasmic volume reaches some threshold, and that either sets off DNA synthesis or (alternatively) inhibits further cell growth? If the extra DNA in Newts and Amphiuma is non-coding "junk", then why are their individual cells larger in exact (?) proportion to the greater amount of DNA per cell?

VI) How do cancer cells disrupt histological structures as drastically as they often do? Is this simply a matter of secretion of proteolytic and other digestive enzymes? Is it a matter of more cell locomotion, in some quantitative sense? Or does this reflect the need for normal homeostatic mechanisms to maintain cell arrangements, such that cells that lose normal behavioral properties undermine the homeostasis?

VII) In the phenomenon of anchorage dependence, what is it about cell elongation (shape per se, tension, state of organization of actin stress fibers, ratio of fibrous actin to diffusible "globular" actin) that permits (or stimulates?) tissue culture cells to pass the checkpoint from G1->S, rather than initiating apoptosis? Why is there such a sharp threshold in the amount of cell spreading (length? area?) needed to satisfy this mechanism? When transformed cells become anchorage independent, what has changed? Conversely, what changes would we need to make in a cell to make it anchorage independent?

VIII) Why is there so much individual variation in people's (patients') responsiveness to any particular form of chemotherapeutic treatment?

IX) Why do people with mutations or deletions in the RB gene only get cancer in their eyes: and why do these cancers arise after many years; instead of arising in all cell types very early in embryonic development? Likewise, when people make transgenic mice with extra copies of some oncogene, then even when dozens of tumors are found early in development, these still represent less than one cell in a million producing a tumor! Why do people accept such results with so little surprise?

X) Occasionally, all too rarely, cancer patients undergo spontaneous remissions: what causes these? Why aren't there any systematic studies of such cases, to find out what happened? Are there several different mechanisms? Variations on some one mechanism? Is the cause the same as it was in the patients that Coley cured with his bacterial extracts?

How to cure cancer by selective initiation of apoptosis:

With minor exceptions (including red blood cells, but I don't know which other cell types; if you find out, please tell me) all the cells of the body contain latent self-destruct mechanisms. Programmed cell death (~= "apoptosis") works by unblocking the active sites of molecules of a special kind of protein-digesting enzyme (caspases) in the cytoplasm. Caspases are "zymogens", in that their active sites are normally/originally blocked by part of their own amino acid chain; but if this part gets ever digested away, then each activated caspase molecule becomes able to digest many other cell proteins, and also become able to unblock the active sites of other caspase molecules in that same cell. This positive feedback self-digestion can quickly digest that particular cell from the inside out.
Please note, however, that apoptosis is not just another word for "cell death"; the word apoptosis refers only to one specific kind of cell death, in which there are certain patterns of DNA digestion, blebbing, shrinkage and rapid inside-out digestion, and there are certain chemicals that stain only apoptotic cells (& I would appreciate it if someone could explain to me how these stains work). Cell death in general is called "necrosis", and it is not unusual for cells to go down fighting. Plenty of cells die without ever setting off this self-digestion mechanism. There are even other kinds of programmed cell death, such as in plants, which self-destruct by different, non-homologous mechanisms. Prof. Dangl in this dept is one of the world's leading researchers on programmed cell death in higher plants, which they use to protect themselves against germs. Note also that lysosomes have nothing to do with apoptosis; it used to be one of the popular theories about tadpole tail destruction, etc. that lysosomes did it; but that turned out to be completely false. Lysosomes are for digestion of molecules within cells; and lysosomes can get out of control and kill cells; but that is different from apoptosis.
All cancer cells contain this latent caspase-based, self-digestion system, just like nearly all normal cells. It is analogous to the self-destruct, radio-activated dynamite charges that NASA puts in all its unmanned rockets, to blow them up if they go off course. Since all cancer cells contain this kind of enzymatic dynamite, and also the equivalents to the radio signalling mechanisms designed to explode it, then a cure for cancer could be invented based on this. In other words, all you have to do to cure cancer would be to figure out some method for selectively initiating this self-digestion ONLY in cancer cells, but not in too many normal cells. That would do the trick! There may be other ways to cure cancer; but I hereby predict that the first one to be discovered will be based on selective initiation of apoptosis. In fact, it has become widely believed that nearly all current drugs for cancer chemotherapy, although they were designed to hurt cancer cells in other ways, all really work by initiating apoptosis (accidentally). For example, cyclophosphamide was designed to bind covalently to guanines in DNA, thereby harming fast-growing cells; and tens or hundreds of thousands of cancer victims have either been cured or put into remission by treatment with cyclophosphamide. But really, what this drug does is to initiate apoptosis, and to do so more in cancer cells than in normal cells. Unfortunately, no one can figure out how or why cancer cells are so much more sensitive to initiation of apoptosis by this or other drugs.
A wide variety of normal functions are accomplished by this same, one mechanism of caspase-based apoptosis. One might have guessed that several different mechanisms would have evolved, either in different kinds of animals or in different tissues, but except for the plants having a different one, animals apparently use the same mechanism for all the following functions.
1* Removal of the tail of tadpoles in metamorphosis. (and certain internal tissues, too!)
2* Removal of tissue between fingers, that otherwise form a web (including in human embryos)
3* Killing of large fractions (up to 90%) of motor and other nerve cells in parts of the spinal cord that have less tissue, and fewer muscles, to innervate.
4* Killing lots of specific cells in nematode cell lineages (I don't have a good sense of why)
5* Killing of viral-infected cells, as a mechanism to minimize spreading of viruses in diseases.
6* "Rejection" of non-self tissue grafts, induced by T-cells. (not really a function, but a byproduct)
7* Poison ivy (also sets off attack of T-cells on "self" tissues. (not really a function, except for ivy)
8** Death of heart muscle cells resulting from heart attacks, and death of nerve cells resulting from strokes and breaking of spinal cord. (Note that this is not a "function", but a tragic by-product of normal mechanisms. If medical methods could be discovered to block apoptosis for a while, then heart attacks and strokes could be made much less harmful!!) (again, not a function but a byproduct)
9?* Clonal selection (elimination) of anti-self B and T lymphocytes. [Direct evidence for this is surprisingly still lacking; Transgenic mice with defective apoptosis develop lupus-like symptoms; and lupus runs in the same families as B-cell lymphoma, both of which make sense if, etc.]

The question then becomes:

#1) What are the different possible ways to set off self-digestion (apoptosis)?

#2) How to make some of the many abnormalities peculiar to cancer cells set off apoptosis?
(So that some of the properties that make them cancerous, or any of the by-products of being cancerous, somehow set off apoptosis just in those cells that have these abnormal properties.)

and maybe #3) How to protect non-cancerous cells from some non-specific initiation of apoptosis?

As an analogy, imagine that we were trying to design an antimissile defense, and we knew that all the incoming missiles would contain the kind of dynamite radio-controlled self-destruct mechanisms that NASA builds into their satellite-launching rockets, even including the radio-control part. That ought to make our job quite a lot easier. It would mean we wouldn't necessarily need to blow up the enemy missiles directly ourselves. It would be sufficient to set off the dynamite charges that all of them already have built into them. We might accomplish that with our own explosives. But there might also be lots of other possible ways to do it. We might even me able to do it purely by radio control. Who knows. And what better subjects for your creative imaginations?

A) In metamophosing tadpole tails (and other tissues), thyroxine initiates the self-destruction; but I don't know what differences between (say) muscle or skeletal cells in the tail, versus muscle or skeletal cells in the main body, cause the former to undergo apoptosis in response to thyroxine while the latter do not. You can cut off parts of the tail and keep them in "organ culture", and they will undergo apoptosis only after treatment with thyroxine. Another bit of evidence is that actinomycin blocks this self-destruction of isolated tails. There seem to be lots of other cases in normal embryonic development where shaping of various organs depends partly on programmed cell death of those cells located at certain locations, sort of "to get them out of the way". For example, a narrow strip of cells between what will become the radius and the ulna (bones) undergoes some form of self-destruction. So do cells along the edge where the two "palatal shelves" come together in mammals to separate the oral cavity from the nasal cavity. These cases have not been well studied by anyone, yet, that I know of.

B) In embryonic nerve cells, one of the popular ideas has been that those cells that do not form muscles to innervate are the ones that die. Dr. Ronald Oppenheim, now at Wake-Forest, but for many years an adjunct professor in the UNC Biology Dept. is the world expert on neural apoptosis.

C) In viral-infected cells, the current paradigm (with a lot of evidence behind it!) is that apoptosis is induced by binding of T-lymphocytes whose T-cell receptor binding sites have exactly the correct shape to bind type I histocompatibility antigens that are holding up a certain peptide, with this peptide being different in different cases and being some kind of breakdown product of a viral protein.

Imagine the following, for the sake of further discussion:
-> Imagine a synthetic chemical that does not itself bind to type I histocompatibility antigens
(or if it does bind to them has a shape not attacked by any of your T-lymphocyte's T-cell receptors)
and imagine that only cancer cells change this chemical into a form that WILL bind to those cells' type I histocompatibility antigens, to form an antigen that will be attacked by T-cells so as to induce apoptosis in the cancer cells (but not in normal cells)!

---> Alternatively, imagine another synthetic chemical that becomes able to activate caspase enzymes, but only acquires this ability because of some change produced in it by some abnormality of cancer cells. In either case, the key point is that cancerousness set off the caspases.

Apparently, cyclophosphamide and other chemotherapy drugs already have this property, except that no one understands how they do it, and therefore cannot improve their ability to do it.
As little as a few % improvement in this mysterious ability to induce self-digestion would make the difference between life and death for tens of thousands of current patients, and for probably 3 or 4 students currently in this class, sometime in your futures. No kidding. That is a million times more likely than that Publisher's Clearinghouse will ever come visit you. So think hard.

 

Wer immer strebend sich bemuht... Den können wir erlosen! Goethe: Faust

 

How to invent new cures for cancer

        SOMETHING IN THIS COLUMN        NEEDS TO BE INDUCED BY
SOMETHING IN THIS COLUMN
Initiation of ApoptosisAbnormalities of cancer cells(alternative means)
i) Caspase activationa) Over-activity of certain kinases
ii) Non-self, viral-like peptidesb) Excessive phosphorylation of certain proteins (held by type I histocompatibility antigens)
iii) Fas/Fas ligand stimulation c) Mutated GTPases, unable to hydrolyse GTP
iv) Other mechanisms?d) Anaerobic metabolism
 e) Inability to halt at cycle checkpoints
 f) Lactic acid production
 g) Secretion of proteolytic enzymes
 h) Disrupted cytoplasmic actin
 i) Abnormal adhesions
 j) Less fibronectin secretion