Hope For Cancer

Gene Therapy simply means artificially changing the genetic information in a cell to alter it’s functioning. The cell may be changed temporarily or permanently. The technology relies upon“genetic engineering” ; that is being able to tinker with the genome. A main problem was how to get foreign DNA into a cell, since they are protected from such large molecules getting in by their cell membranes. In the 1950′s it was found that genetic information could sometimes be transfered from one cell to another by soaking them in a solution of calcium chloride, which opened up “pores” within the cell membrane. Another method was “electroporation”, meaning giving a small electrical shock to the receiving cell to increase it’s membrane permeability. The problem with these techniques was the difficulty in transfering large pieces of DNA, and how to get the new DNA into every cell. Other challenges including getting the DNA specifically into the cells we wanted to treat, getting the new gene(s) into the right place in the “transduced” cell, and getting the new gene(s) to turn on (and stay turned on!). Sometimes the unhealthy human cells were removed from the patient and the DNA transfer was done in the laboratory, a technique called “in vitro” (in glass). The cells that has successfully taken up the new DNA could be isolated and then re-injected back into the patient. The other option was to do the gene transfer within the patient themselves, by directly injecting the with the new DNA carried by a “vector” . The most effective vectors were found to be stripped-out viruses, since these naturally bound to human cells and injected their foreign DNA into them. Placing the new genes directly into the patient is called“in vivo” (in life) gene therapy.

What is Gene Therapy Cont.

It was found that viruses were constructed out of a protein coat surrounding a small amount of DNA. The virus had an actual injection system whereby it could land on a host cell (it has little “legs” making it look like a lunar lander) poking a hole with a hollow tube through the cell’s membrane. Once “docked” onto the host cell, the viral DNA is injected into it. The DNA may just float about in the cell fluid (“cytoplasm”) or may insert itself into the host DNA. Empty viral protein “capsules” could be stuffed with custom DNA and let loose to transduce this DNA into the target cell. Furthermore, certain types of viruses had a predilection to latch on to particular body cells. For example, adenovirus tends to go the lung while hepatitis virus attatches to liver cells. Thus, we could get at least some “specificity” to correctly target the modified viral DNA to the diseased cells.

A further way of getting new gene information into cells using viruses is by linking the information to a class of viruses called “retroviruses”, which include the deadly HIV (AIDS causing) virus as well as less harmful mouse (“murine”) viruses which can be used for therapy. Transfering genetic material this way is called “In Vivo Gene Therapy”, meaning instilling the gene via a “living” entity directly into the Human body. The gene we want to transfer can be “spliced” into the retrovirus, which has the ability to enter a Human cell and integrate directly into the Human genome! This exact capability which makes retroviruses so dangerous also makes them extremely useful tools for gene therapy, as we will see later with specific examples.

On recent idea of a way to get a new gene into a cell is by encapsulating them in a “liposomes”, which are basically storage sacs which float about in the cell cytoplasm to store fats, enzymes, proteins, drugs, or whatever. They appear under the light microscope as little “bubbles” within the cell fluid, and have the ability (sometimes) to pass through the normally poorly permeable cell membranes. Cell membranes have to be very selective in what they let through, otherwise they would serve no purpose in protecting the cell from the hostile outside world. Apparently, things which ordinarily would be too large to pass through cell membranes can be packaged in real or artifical liposomes, and then induced to enter cells. Once inside, the liposome coating may be broken down by intracellular enzymes, and the contents (such as a new gene) released. It is possible (but far from certain) that the new gene will successfully integrate into the “host” DNA, and start functioning! The chances for this happening are greater if many “copies” of the gene are introduced into the host cell, instead of just one or a few . Much research is going on using liposomes as the transporter.

An important development was the ability to isolate the particular genes we wanted
from a genome which might contain 3000 genes! It was easier to get a small bit of foreign genetic material to penetrate through a receiving cells membrane using calcium chloride or electroporation, than to try to get a large chromosome through. Also, it was much easier to pack a small piece of genetic material into an empty virus shell than to incorporate a large piece. Furthermore, we didn’t want to transfer unwanted genes into our “host” but only ones coding for the specific attribute of interest. The ability to “map” genes to particular chromosomes, and then to actual physical areas on the chromosomes, was crucial to isolating, cutting out and transfering genes. The actual method of localizing genes was called“hybridization”, whereby we would work backward from the product that the gene made, looking in the cell for the“messenger RNA” coding sequence that was coming out of the nucleus to attatch to the cell’s ribosomes (in the cytoplasm). These ribosomes represented the actual “machinery” which translated the genetic message into protein and enyzmes which in turn build a living organism. By examining the RNA, we could elucidate the corresponding DNA sequence that producing it. By synthesizing a radioactively labelled “complimentary” DNA sequence and putting it into the cell, we could see where it migrated and attatched to within the nucleus, and viola, know where the gene of interest was located.

Once the gene of interest was localized, we had to know how to “cut it out” (excise it) so as to isolate it. Fortunately, we discovered that the cell itself produced special enzymes to slice and dice up DNA, and that we could use these enzymes to cut up the DNA for our own purposes. These particular enzymes were called “Restriction Endonucleases”, and more are still being discovered today. They acted as “scissors” for cutting the DNA only in a specific area. Now the DNA is composed of four “bases”, which are Adenine, Guanine, Cytosine and Thymine. Adenine always pairs to Guanine on the “complementary strand” (the opposing strand of the double helix), while Cytosine always pairs to Thymine. It is the varying arrangement of these these 4 simple bases which gives DNA it’s final complexity, and which accounts for the innumberable possibilities– complexity is from the simple, repeated over and over!

The Restriction Endonucleases “cleave” the DNA at a particular site, such as when a Cytosine follows two consecutive Adenines which themselves follow a Guanine. There may be many such sites in a long DNA molecule, and so several Restriction Endonucleases which cut in different areas may be used to isolate a sequence of interest. Even after cutting the DNA up, we still must somehow tease out the piece of it we want. Since the pieces of DNA will have different lengths, they will also have different weights and we can separate them by using an electrical current to drag them along a “medium”, like filter paper or special “agarose gel”. The heavier pieces will move more slowly than the lighter ones, and so can distinguish them by weight (and thus by length). Once collected, we are now ready to put our piece of DNA, with it’s particular genes(s), into a receiving“host” cell. This is called“Gene Transfer”, and results in a new “combination” of genes of genes in the host. Thus the whole process is called “Recombitant DNA Technology”.

The initial uses for this new “Recombitant DNA Technology” was in putting genes that manufactured specific proteins or enzymes into bacteria, to make useful chemicals in large quantities. For instance, it takes many pig (“porcine”) pancreas’ to extract enough purified insulin for diabetics, but simple bacteria (like the E.Coli found in the human intestines) can be modified by insertion of the insulin-producing gene to become factories manufacturing insulin. These bacteria can be grown in large cultures (glass dishes), and the insulin they produce drained off, packaged, and sold to diabetic patients. It turns out to be a cheaper, safer way to make insulin, since there are no foreign pig proteins contaminating it (which can cause allergic reactions). Human Growth Hormone used to be extracted from thousands of dead people’s brain pituitary glands, and was very expensive. It’s a needed hormone for children who lack it, since they will be midgets without it. Recombitant DNA Technology allowed the gene that “codes” for the production of growth hormone to be isolated, cut out of the neighboring DNA, and transferred into bacteria. These bacteria can then produce great quantities of Growth Hormone at much less cost and logistical problems than collecting it from deceased people’s brains. A further use was developing “Oil Eating” bacteria to combat oil spills from tankers. Thus, the basic technology of “Gene Transfer”, at least into bacteria, is already well established and used every day .

The next logical step was using Recombitant DNA Technology in Human cells instead of just bacteria. The idea was to insert a gene to make a necessary enzyme in human cells that were missing them. The lack of an enzyme, caused by a flaw in or absence of necessary genes, is responsible for many “metabolic” diseases. While these diseases are mostly rare, they are often severe or fatal for those afflicted. Examples include Cystic Fibrosis and Adenine Deaminase Deficiency . If we could insert the proper genes into cells lacking them, the crucial enzymes would be produced, and these diseases cured. This was the first real attempt at primary “Gene Therapy”, but proved to be a lot harder than working with bacteria. For instance, it is more difficult to get the new genes into human cells, and to have them “integrate” into the right place in the human genome. With bacteria, we can bombard them with the new genes, see which bacteria take them up and separate them out of the bunch. Then we can induce them to divide to produce new bacteria with the desired gene, and grow them in vast quantities. Trying to put genes into Human Cells is tougher, since we can’t just “select out” the cells which have uptaken the new gene, or cause them to divide and grow more than they ordinarily would. Also, even after we get the new genes into the Human cells, there is no guarantee that they will migrate to the right place in the genome to function properly; they might just lie dormant and do nothing.

Furthermore, even when they do go to the right place, and start working, their action (called“expression” ) may be temporary. For reasons we are still trying to figure out, they may get turned off and quit working, defeating the whole therapy. Moreover, there are safety issues. it is possible that inserting the new gene into the diseased cells will actually turn on an Oncogene or turn off a Suppressor Gene, starting a cancer! Also, if we use “living” retroviruses to transfer genes, they may mutate into dangerous forms capable of “replication”, and actually start a new disease in the Human population! In general, however, this risk is small and the possible benefits of inserting functioning genes into gene-lacking cells cells are great– curing otherwise deadly diseases. The first success has already been seen with this therapeutic gene transfer, for the Adenine Deaminase Deficiency Syndrome.

When the Adenine Deaminase gene is successfully integrated into a diseased child’s genome, the disease is cured. Cystic Fibrosis cells have been “cured” in Clinical Trials, and future possibities include cure of all sorts of hereditary genetic diseases, from hemophilia and Tay Sachs disease to Muscular Dystrophy and Parkinson’s Disease.

The most complex use of Gene Therapy is going beyond replacing a non-functioning gene with a working one– it is actually turning on or off existing genes within Human Cells, or even fixing damaged ones. We are currently mapping the entire Human Genome, consisting of about 3 billion base pairs, to localize the genes for every physical human trait- – eye color, height, fingerprints, inborn diseases, etc. This enormous undertaking will give us a “road map” of all Human genes, enabling us to quickly identify missing, broken or duplicate ones in patients. When we can go into cells and modify the genes at will, we shall have incredible powers to cure disease– and“genetically engineer” ourselves. For example, we will be able to change a person’s height or eye color, cure Down’s Syndrome and maybe regrow amputated limbs or weak organs. We shall likely be able to increase our lifespan by turning off “aging genes”. We will be able to cure most every illness, including any cancer, by merely shutting off the “grow” message in sick cells, and encouraging the healthy cells to grow instead . The possibilities are limited only by our imagination. The usage of them, for good or evil, will surely test the basic nature of humankind.

This is just an excerpt from our Complete Cancer Treatment Transcript. Much more, including latest treatments, can be sent to you by email when you order the complete transcript at a nominal cost.

Obviously, chemotherapy works by killing cancer cells. In our current theory, it seldom, if ever, kills the last remaining cancer cells. Instead, it dramatically reduces the number of such cells, and the body’s immune system “mops-up” those few that remain. This is a paradox, since many chemotherapy agents weaken the immune system, thus compromising it’s ability to recognize and kill abnormal cells. Various agents kill cancer (and normal) cells in different ways, and it is instructive to get a better understanding of this process.

The study of how chemotherapy effects living cells is called molecular biology and this field has exploded with new information in the last 2 decades, as nuclear physics did in the early twentieth century under Einstein. To understand how chemotherapy effects both normal body tissues and cancers, we look at living organisms at their”cellular” level. All living things have as their basic unit the “cell” ; similar cells combine to form “tissues”, and tissues combine to form “organs” . This is analogous to the way in which atoms are the basic unit of elements, elements of molecules, and molecules of compounds. Simple creatures may have only a single cell (e.g. a bacterium or amoeba), while plants, animals and humans are composed of billions or trillions of cells. A spherical piece of flesh 1/2 inch across contains about a billion cells! In our bodies old or injured cells die, while new ones form – this constant process is crucial to continued life. We know that if we give enough radiation to any cell, it will die. To appreciate how chemotherapy works, however, we must look even deeper (smaller) than the cell, at it’s “subcellular” components.

Individual cells were first observed in the 17th century by Leiwenhook, who had invented the microscope. More powerful magnification showed a world of activity going on within cells, the process of “life” . Electron microscopes now show yet more.

The first thing noted was that every mammalian cell had a “membrane” around it, a darkly staining central spot called the “nucleus”, and between the outer membrane and the inside nucleus was “cytoplasm”fluid. Closer inspection showed the cytoplasm to actually be filled with apparent machinery, called”organelles” for small organs. The nucleus was made up of dark staining strands, called”chromosomes”. These chromosomes became especially visible when the cell divided, a process called”mitosis” . Furthermore, each chromosome was a slightly different shape, but they appeared to arrange into pairs at mitosis. Closer study of the chromosomes showed that there were 48 total in a humans, 23 which paired up as “autosomes” and 2 “sex chromosomes” . In females both “sex chromosomes” were called “X-chromosomes” (for their shape), while males had one “X-chromosome” and a smaller”Y-chromosome” . Staining and studying the chromosomes during mitosis was called a “karyotype”, and it was soon seen that various serious diseases corresponded to abnormal chromosome patterns . For example, in Down’s syndrome where severe mental retardation and abnormal features were present at birth, these children were shown to have three Chromosome #21′s (“trisomy 21″) instead of the normal two. In girls who had short stature, webbed neck, and were infertile, they were found to be missing one of their two sex chromosomes (“Turner’s syndrome”). Many such syndromes were found, but not every obviously inherited disease had clearly abnormal chromosomes.

It became obvious that chromosomes controlled heredity, and that one of each pair of chromosomes was inherited from each parent. Chromosomes themselves were found to be composed of thousands of much smaller elements called”genes”, short for “genetic materials”. Somehow, a”genetic code” existed that told the cell how to live, function, and even when to die. This code was “cracked” (to a point) in the 1950′s by Watson and Crick, who demonstrated the model for “Deoxyribose Nucleic Acids” (“DNA”) . These DNA molecules were shown to be twisted into a “double helix” which formed the genes. DNA itself was shown to be made up of a long “sugar” backbone (the “ribose”) and just four other molecules,(the “nucleic acids”). These four molecules (adenine, cytosine, guanine, and thiamine) were paired up on the two “strands” forming each double helix – adenine always paired to thiamine, while guanine always paired to cytosine. The amazing thing was, that the arrangement of these 4 molecules were different in every different gene, made up the genes, and so would determine every physical characteristic of every plant, animal and human! There were found to be about 3 billion “DNA base-pairs” in the human”genome”, different for everyone except identical twins. It was soon seen that damage to these ultramicroscopic (smaller than an ordinary microscope could see) base pairs were associated with every inherited disease known. For the cell to produce new products (“proteins”) the DNA double-helix “unzipped”, and a strand of “messenger RNA” was formed along one DNA strand. This RNA stand then separated from the parent DNA, and traveled outside the nucleus to the cytoplasm. In the cytoplasm exist protein manufacturing factories, called “ribosomes”, which get their message on what do do from the messenger RNA. Proteins and enzymes are then produced, which may utilized inside the cell, or sent outside of it as a “gene product” such as a hormone or antibody. Now we knew that if this process went awry, and DNA was damaged, cell products would be abnormal and disease could result.

One more important facet before describing chemotherapy effects is a deeper understanding of how cells divide. When the cells divided, the double helix of DNA base pairs “unzipped” and doubled itself by forming two new “complimentary” strands, using the two previous strands as a “template” . As mentioned, this process, called “mitosis” for regular body cells, is essential for life, but also for cancers to grow and spread. We know that we all start out in womb life from the contribution of a sperm from our father and an egg (“ovum”) from our mother. To form these “germinal cells” in our father’s testicles and mother’s ovaries (our parents “gonads”), a unique type of cell division occurred, called”meiosis” . For normal cell division, mitosis, the DNA duplicates (doubles), the divides in half, so we end up with 2 identical “daughter” cells(which can each go on to double their DNA and divide again). Each of these daughter cells still retains the contribution of DNA design from both parents, even if the individual is 100 years old. Thus, there is an identical type, and amount, of DNA in every body cell – each cell has within it the information on how to form a whole new body! Now if each parent gave us this type of cell, with a full amount of DNA, we would have enough information for two bodies – not one. Therefore, the “meiosis” process that occurs to make sperm and eggs cuts the amount of DNA in half, instead of ultimately keeping it the same like mitosis. Fascinatingly, the DNA is sliced in half differently for each sperm or egg produced, which explains why siblings look different. As will be seen, the testicles and ovaries are particularly sensitive to chemotherapy damage, and if they are “overdosed” then infertility (“sterility”) will result.

The sperm and egg combine upon the spongy inner lining of the uterus (“endometrium”), re-forming the normal amount of human DNA in this newly “fertilized egg”. From this point on, until the incipient child begins forming their own eggs or sperm at puberty, all cellular division is mitotic, not meiotic. Thus, the normal human compliment of DNA, one-half being from each parent, is restored in every cell division. The fertilized egg starts dividing, forming an”embryo” . At first, all of the cells are the same (“pleuripotential”), but then the genes within some of the cells activate and cause them to change (“differentiate”) from the other cells. Thus muscle, fat, heart, lung, bone, brain, skin etc. form, from these specialized cells. After 8 weeks, the embryo has a heartbeat and is recognizable as a tiny human, and is called a”fetus” .

From the fetal point onward, all the organs are formed, and the merely develop and grow larger.
In womb life, early childhood, and through puberty all of the body’s cells are “turned on” to divide and grow an adult human. The genes exert very exacting control over cellular division, to ensure that it does not run amok. Gradually, certain systems become fully grown, and cell division completely ceases. Other systems will regenerate new cells to replace those that have died as a result of old age or injury, while still others constantly generate new cells throughout life. For example, the brain cells (“neurons”) cease dividing by puberty, and will never divide again in a normal brain. The cells of the liver or skin are capable of dividing to replace injured ones, while the blood cells and intestinal lining are continuously being renewed. As long as tight control is maintained by the genes, everything grows in it’s proper time.

Each of the body’s cells has a specific “cell cycle” related to reproducing, and this cycle may change over time. The cell cycle, and thus division, is controlled by the genes. A cell may spend a prolonged period (or even the rest of it’s existence) in a quiescent period, where it is not reproducing (the “G1 phase”). If the genes trigger instructions for a cell division, the cell starts duplicating it’s DNA (the “S phase”). Once the DNA is doubled, it prepares to divide (the “G2 phase). Then the actual division takes place (the “M phase”) to produce two identical daughter cells. At certain points in the cell cycle, there are “checkpoints” to ensure that the DNA is intact, that is has doubled normally, and that the cell is indeed ready to divide. Each of these division checkpoints is controlled by genes, which should not let the division take place is something is wrong. Normally, this system works with incredible speed and harmony.

Now we have a background to understand what happens to make a cell turn cancerous . Something damages the genes that control cell division, resulting in a cell which divides out of control. That something may be a chemical (“carcinogen”), virus, radiation, or just a random”mutation” (change; deviation) that occurred during a previous division. Anything that damages the controlling genes in a cell can lead to cancer . The genes damaged may be the ones that “check” the cell at the division checkpoints, and so erroneously allow a damaged cell to divide. Alternatively, they may be the same genes that were normally turned on in the womb and childhood, but in adulthood they should be turned off (“oncogenes”) . Another scenario is the damaged genes are ones that normally suppress excessive division (“suppressor genes”) and now the cell divides without regulation. Whatever genes were damaged to cause cancer, it is ultimately a disease of the DNA, the molecules which form the genes. Chemotherapy can damage DNA or interfere with the protein products it produces, either killing a cell or causing it to become abnormal.

At the cellular level, then, chemotherapy either blocks something needed for the DNA to replicate (preventing cell division), or interferes with protein production by disturbing the RNA, ribosomes or their necessary “metabolites”. Since both division and protein production are essential to a cell’s functioning, derailing either effectively kills that cell. In cancer terms, a cell which can’t divide is as good as dead, for it can no longer add to the “tumor burden”. Interestingly, some cells produce proteins which act as local hormones and stimulate their own, or their neighbors division – these are called “autocrine” proteins. These are well described in brain cancer (“gliomas”). Being able to block these autocrine substances will naturally slow cancer replication. Of course, effectively blocking cancer cell division will also impair normal cell division, leading to side effects. This is less problematic when bacteria are treated with antibiotics, since the bacterial ribosomes are different than human (and animal) ribosomes. Therefore, bacterial reproduction can selectively be blocked by targeting those non-human ribosomes, leaving the human ones untouched. The problem in giving chemotherapy is that the DNA, RNA, Ribosomes and major Proteins within cancer cells are virtually the same as in normal cells! Of course, we said their are differences, for the DNA is damaged and abnormal RNA and proteins may be made. However, the differences are slight, often only at the gene level, and our current agents don’t distinguish normal cells from cancer cells at that level. The crude way we do distinguish normal cells from cancer cells is by the rate of cell division, which tends to be faster in cancer cells. Thus, depriving the cells of what they need to divide, or otherwise poisoning the division process, will tend to selectively weed out cancer cells first . Naturally, quickly dividing normal cells (i.e. blood cells, scalp hair follicles, gastrointestinal lining cells) will also succumb, explaining the classic side effects of chemotherapy. However, they can repair damage and heal better also.

The previous discussion also explains the paradox of why more aggressive cancers may actually be more easily cured than less aggressive (“indolent”) ones. Aggressive cancers (e.g. choriocarcinoma, lymphoblastic lymphoma, small cell lung cancer) tend to be quickly dividing and so rapidly killed off by chemotherapy. In contrast, more indolent cancers (e.g. low grade sarcomas, chronic lymphoma, hormone-responsive breast cancer) that are slower growing won’t be killed off much faster than slowly cycling normal cells (i.e. muscle, nerve, fat, bone) and so the chemotherapy will hit the normal cells just as hard. This causes the side effects of the required doses to kill off most all of the cancer cells too hard for the body to tolerate. Enough of the chemotherapy would decimate the cancer, but the patient would succumb also. Thus we are relegated to giving lower doses, which may (or may not) effectively kill enough cancer cells to make a noticeable(“clinical”) difference.

Interestingly, their is a range of sensitivities in the tumor cells (even a single patient in a single tumor). Some will be readily killed by the chemotherapy, but others will live. Even if 95% of the cancer cells are killed by the drug, the 5% remaining tend to be more resistant and require much more drug to kill them, probably more than the normal cells can tolerate. This is called the “Goldie-Coleman” hypothesis – as we are more successful in killing cancer cells, the remaining ones are the most impervious to our treatment . We need to kill the vastest majority right at the outset, since the resistant population can develop over time (much like insects grow resistant to pesticides). This is a major reason for using multiple drugs, and for using multiple therapies such as radiation and surgery together with chemotherapy, instead of waiting until the cancer grows back to try other therapies.

This is just an excerpt from our Complete Cancer Treatment Transcript. Much more, including latest treatments, can be sent to you by email when you order the complete transcript at a nominal cost.