**3. What causes cancer?**

Short answer is exposure to radiations, chemical pollutants like smoking, viral infection, or genetic inheritance. This is the most important question and I want to spend more time explaining the causes of cancer. This is where we spent most of the \$30 billion during the lasts 30 years trying to understand how normal cells become abnormal. If we understand how they become abnormal, we should be able to treat them.

In 1971, President Richard Nixon declared war on cancer and released hundreds of millions of dollars for cancer research. He challenged Americans, the way President John Kennedy had challenged Americans a decade earlier to land men on the moon and bring them back safely. Although both presidents had great ideas, but there was a major difference.

our ears, our arms, and our legs. Although each cell carries complete instructions to make all the organs, not all cells make all organs, but each cell begins to receive specific instructions to take a different role as it begins to make more copies. We call this process the cell differentiation.

The Impact of Sequencing Human Genome on Drug Design to Treat Oral Cancer

http://dx.doi.org/10.5772/intechopen.80231

37

Damage to DNA nucleotide called mutations produces disastrous change in the information molecules. As I said above, mutations are caused by exposure to radiations, chemical pollution, viral infection, or genetic inheritance. In addition to hundreds of chemicals isolated from tobacco tar, the most potent carcinogen is nicotine N-oxide. As mutation begins in a single biological molecule, it is called a point mutation. To study changes in genetic profile of a single cell, we examine the entire genome of the same single cell. As cells grow rapidly, other mistakes in DNA replications are most likely to occur such as deletion, insertion, or inversions of nucleotide sequence. Such additional mutations are responsible for causing major diseases. Before the completion of Human Genome Project, NCI screened thousands of chemicals, plant extracts, and animal extracts for their antitumor activity. By trial and error, one in several thousand turned out to be useful. There was a need to make a rational approach to design drugs.

In 1990, United State Congress authorized 3 billion dollars to NIH to decipher the entire human genome within 15 years that is the total genetic information that makes us human called the Human Genome Project. Thousands of scientists from 6 industrialized nations and 20 biomedical centers joined our effort, and within 13 years, the entire human genome was deciphered and published in the scientific journal *Nature* [4–8] and linked to website. If you

On April 3, 2003, we read the Human Genome, the entire book of life. We found that less than 2% of the genome codes for proteins and the rest is the noncoding region which contains switches to turn the genes on or off. We can cut and paste genetic letters in the noncoding region which could serve as markers and which has no effect, but a slight change in the coding

A single cell is so small that we cannot even see with our naked eyes. We have to use a powerful microscope to enlarge its internal structure. Under an electron microscope, we can enlarge that one cell up to nearly a million times of its original size. Under the electron microscope, a single cell looks as big as our house. There is a good metaphor with our house. For example, our house has a kitchen, the cell has a nucleus. Imagine for a moment that our kitchen has 23 volumes of cookbooks which contain 24,000 recipes to make different dishes for our breakfast, lunch, and dinner. The nucleus has 23 pairs of chromosomes which contain 24,000 genes which carry instructions to make proteins. Proteins interact to make cells; cells interact to make tissues; and tissues interact to make an organ and several organs interact to make a man, a mouse, or a monkey. In every cell of our body, we carry 16,000 good genes, 6000 mutated genes responsible for 6000 diseases, and 2000 pseudogenes that have lost their functions, during evolutionary time. Our entire book of life is written in four letters, and they are A (adenine), T (thymine), G (guanine), and C (cytosine). These four chemicals are called nucleotide and they are found in the nucleus of all living cells including humans, plants, and animals. Instruction in a single

have an access to a computer keyboard, you have access to all that information.

region makes a normal cell abnormal or cancerous.

**3.1. Mutations**

At the time President Kennedy made that famous speech in the US congress, most of the engineering problems had already been worked out. For example, we already knew the engine thrust and its lift off power needed to leave Earth's gravity. It was calculated to be 7 miles/minute or burning fuel in the absence of air to excel the spacecraft. These engineering problems had already been solved and the knowledge was already available. Only money and trained men power were needed to build the spacecrafts. Within 10 years of that speech, President Kennedy's dream was turned to reality. On July 20, 1969, Americans landed men on the moon and brought them back safely.

But when President Nixon declared war on cancer, money was made available, but the knowledge was not there. We did not exactly know the inner working of a single living cell and how a normal cell functions, and we did not know why the normal cell becomes abnormal or cancerous. Some basic knowledge was available. In 1953, the big discovery was made in Cambridge University, England. Crick and Watson had determined the double helical structure of the genetic material DNA and postulated how the living cells divide and cells grow, and they were awarded Nobel Prize for their discovery [3]. Armed with this knowledge, we were ready to understand how a normal cell functions. Crick and Watson also opened the doors for the Nobel Prize Club. Every year, since then, a new Nobel Laureate was added to the genetic club. Soon after, Marshall Nirenberg broke the genetic code and unlocked the secret of life by showing that only three nucleotides code for an amino acid, the building block of protein. The remaining codes were deciphered by Salvador Ochoa, followed by an Indian Scientist, Govind Khorana who shared the Nobel Prize with Walter Gilbert.

After President Nixon's speech, it had taken about 20 years to understand how a normal cell functions and how it becomes abnormal by exposure to radiations, chemical pollutants, viral infection, or genetic inheritance. Let me summarize below the work of a dozen Nobel Laureates: To understand cancer, you have to understand how a normal cell functions and how it becomes abnormal. We made step by step progress over the past 30 years. First, you might ask why we study a single cell and why a single cell is so important. The fact is our life begins with a single cell. You and I are the loving union of our parents. Both parents contribute half the genetic material to each cell. Our father contributed one sperm and our mother contributed one egg. When the egg and sperm join together, we were conceived. We grow as a single cell in our mother's womb. This single cell has a set of complete instructions to construct us within in 9 months. By the time we are grown up to adulthood, that single cell makes over 100 trillion copies of itself.

During replication, if we introduce slight error in the nucleotide sequence called mutation by exposing to radiations or chemical pollutants including nicotine, viral infection, or genetic inheritance, the error is copied in every other cell. At this stage, pregnant mothers should be extremely careful what they eat and what they drink and to avoid exposing the fetus from the secondhand smokers and should stay away from smokers as far as they could. Every cell in our body has a complete library of our genome and carries complete instructions to make our brain, our nose, our ears, our arms, and our legs. Although each cell carries complete instructions to make all the organs, not all cells make all organs, but each cell begins to receive specific instructions to take a different role as it begins to make more copies. We call this process the cell differentiation.

### **3.1. Mutations**

In 1971, President Richard Nixon declared war on cancer and released hundreds of millions of dollars for cancer research. He challenged Americans, the way President John Kennedy had challenged Americans a decade earlier to land men on the moon and bring them back safely.

At the time President Kennedy made that famous speech in the US congress, most of the engineering problems had already been worked out. For example, we already knew the engine thrust and its lift off power needed to leave Earth's gravity. It was calculated to be 7 miles/minute or burning fuel in the absence of air to excel the spacecraft. These engineering problems had already been solved and the knowledge was already available. Only money and trained men power were needed to build the spacecrafts. Within 10 years of that speech, President Kennedy's dream was turned to reality. On July 20, 1969, Americans landed men on

But when President Nixon declared war on cancer, money was made available, but the knowledge was not there. We did not exactly know the inner working of a single living cell and how a normal cell functions, and we did not know why the normal cell becomes abnormal or cancerous. Some basic knowledge was available. In 1953, the big discovery was made in Cambridge University, England. Crick and Watson had determined the double helical structure of the genetic material DNA and postulated how the living cells divide and cells grow, and they were awarded Nobel Prize for their discovery [3]. Armed with this knowledge, we were ready to understand how a normal cell functions. Crick and Watson also opened the doors for the Nobel Prize Club. Every year, since then, a new Nobel Laureate was added to the genetic club. Soon after, Marshall Nirenberg broke the genetic code and unlocked the secret of life by showing that only three nucleotides code for an amino acid, the building block of protein. The remaining codes were deciphered by Salvador Ochoa, followed by an Indian

After President Nixon's speech, it had taken about 20 years to understand how a normal cell functions and how it becomes abnormal by exposure to radiations, chemical pollutants, viral infection, or genetic inheritance. Let me summarize below the work of a dozen Nobel Laureates: To understand cancer, you have to understand how a normal cell functions and how it becomes abnormal. We made step by step progress over the past 30 years. First, you might ask why we study a single cell and why a single cell is so important. The fact is our life begins with a single cell. You and I are the loving union of our parents. Both parents contribute half the genetic material to each cell. Our father contributed one sperm and our mother contributed one egg. When the egg and sperm join together, we were conceived. We grow as a single cell in our mother's womb. This single cell has a set of complete instructions to construct us within in 9 months. By the time we are grown up to adulthood, that single cell makes over 100 trillion copies of itself. During replication, if we introduce slight error in the nucleotide sequence called mutation by exposing to radiations or chemical pollutants including nicotine, viral infection, or genetic inheritance, the error is copied in every other cell. At this stage, pregnant mothers should be extremely careful what they eat and what they drink and to avoid exposing the fetus from the secondhand smokers and should stay away from smokers as far as they could. Every cell in our body has a complete library of our genome and carries complete instructions to make our brain, our nose,

Although both presidents had great ideas, but there was a major difference.

Scientist, Govind Khorana who shared the Nobel Prize with Walter Gilbert.

the moon and brought them back safely.

36 Prevention, Detection and Management of Oral Cancer

Damage to DNA nucleotide called mutations produces disastrous change in the information molecules. As I said above, mutations are caused by exposure to radiations, chemical pollution, viral infection, or genetic inheritance. In addition to hundreds of chemicals isolated from tobacco tar, the most potent carcinogen is nicotine N-oxide. As mutation begins in a single biological molecule, it is called a point mutation. To study changes in genetic profile of a single cell, we examine the entire genome of the same single cell. As cells grow rapidly, other mistakes in DNA replications are most likely to occur such as deletion, insertion, or inversions of nucleotide sequence. Such additional mutations are responsible for causing major diseases. Before the completion of Human Genome Project, NCI screened thousands of chemicals, plant extracts, and animal extracts for their antitumor activity. By trial and error, one in several thousand turned out to be useful. There was a need to make a rational approach to design drugs.

In 1990, United State Congress authorized 3 billion dollars to NIH to decipher the entire human genome within 15 years that is the total genetic information that makes us human called the Human Genome Project. Thousands of scientists from 6 industrialized nations and 20 biomedical centers joined our effort, and within 13 years, the entire human genome was deciphered and published in the scientific journal *Nature* [4–8] and linked to website. If you have an access to a computer keyboard, you have access to all that information.

On April 3, 2003, we read the Human Genome, the entire book of life. We found that less than 2% of the genome codes for proteins and the rest is the noncoding region which contains switches to turn the genes on or off. We can cut and paste genetic letters in the noncoding region which could serve as markers and which has no effect, but a slight change in the coding region makes a normal cell abnormal or cancerous.

A single cell is so small that we cannot even see with our naked eyes. We have to use a powerful microscope to enlarge its internal structure. Under an electron microscope, we can enlarge that one cell up to nearly a million times of its original size. Under the electron microscope, a single cell looks as big as our house. There is a good metaphor with our house. For example, our house has a kitchen, the cell has a nucleus. Imagine for a moment that our kitchen has 23 volumes of cookbooks which contain 24,000 recipes to make different dishes for our breakfast, lunch, and dinner. The nucleus has 23 pairs of chromosomes which contain 24,000 genes which carry instructions to make proteins. Proteins interact to make cells; cells interact to make tissues; and tissues interact to make an organ and several organs interact to make a man, a mouse, or a monkey. In every cell of our body, we carry 16,000 good genes, 6000 mutated genes responsible for 6000 diseases, and 2000 pseudogenes that have lost their functions, during evolutionary time.

Our entire book of life is written in four letters, and they are A (adenine), T (thymine), G (guanine), and C (cytosine). These four chemicals are called nucleotide and they are found in the nucleus of all living cells including humans, plants, and animals. Instruction in a single gene is written in thousands of AT/GC base pairs that are linked together in a straight line and we call them DNA (deoxyribonucleic acid—Nobel prize was awarded to Crick and Watson for describing the double helical nature of the DNA structure). When thousands to millions of AT/GC base pairs contain information to make a single protein, we call that portion of AT/GC base pairs a gene (Nobel Prize was awarded to Khorana and Gilbert for making a functional gene). The genes begin to function with a start codon (AUG) and stop working at the following three stop codon: UGA, UGG, and UAG. After the stop codon, no more amino acids are added and DNA synthesis stops. If we count all the AT/GC base pairs in a single cell of our body, we will find that there are 3.2 billion pairs present in every cell. The entire AT/ GC sequence of 3.2 billion base pair is called the human genome or the book of our life which carries total genetic information to make us.

example, millions of years ago, humans and dog shared some of the same ancestral genes; we both carry the same olfactory genes. Since humans do not use these genes to smell for searching food, these genes are broken and they lose their functions in humans, but we still carry them. We call them pseudogenes. Recently, some Japanese scientists have activated the pseudogenes; this work may create ethical problem in future as more and more pseudogenes

The Impact of Sequencing Human Genome on Drug Design to Treat Oral Cancer

http://dx.doi.org/10.5772/intechopen.80231

39

The above DNA nucleotide bases constitute the genetic map of the normal human being; what makes them abnormal and makes us sick is the mutation in the coding regions of the genome. Now, we can examine the tumor genome of the oral cancer patients to identify specific mutations responsible for causing the disease. As I said above, less than 2% of the genome codes for amino acids. Slightest damage to the coding regions of the four nucleotides A, T, G, and C either by radiations, chemical pollution (from tobacco tar), genetic inheritance, or viral infection or by insertion, deletion, or inversion of the nucleotide bases code for wrong or abnormal

Although you and I are both human and yet no two individuals look the same because the AT/GC base pairs in each of us are arranged slightly differently, a difference of one nucleotide in a thousand base pair. The amazing fact is that out of 3,000,000,000 AT/GC base pairs, only 3 pairs of AT/GC code for a single amino acid, the building blocks of protein, called a codon (Nobel Prize was awarded to Nierenberg and Ochoa). Codons are the most important collections of AT/GC base pairs because they have correct instructions to make the right amino acids (there are only 20 amino acids; they randomly combine to make a protein). Thousands of amino acids make a protein and thousands of proteins make a cell; billions of cells make a tissue and hundreds of tissues make an organ and several organs make an individual such as

As I said above, the old cells begin to die and they are constantly being replaced by healthy cells. Why do the normal cells become abnormal or become cancerous? Any factor that disrupts the 2% of the coding region of our genome will alter its function by slightly altering its code; an altered codon will code for a wrong amino acid and a wrong amino acid will give a wrong protein and it will make normal cell abnormal. When the functions of codons are disrupted intentionally or unintentionally, we alter the codon's function. For example, intentionally we alter a codon by smoking and unintentionally by exposure to environmental pollution such as chemicals or radiations. Altered codons have wrong information to make wrong amino acids. Wrong amino acids make wrong proteins and wrong proteins make wrong cells and wrong cells grow much faster than the normal cells and become abnormal or

Four factors will disrupt a codon's function. Two are minor such as viruses and inherited oncogenes, and two are major such as radiations and chemical pollutants in our environment.

HPV causes more than 32,000 cases of cancer including oral cancer every year in the US. It is also very preventable by giving HPV vaccine for children at ages 11–12 which can protect them.

you and me. This is how normal cell functions and we begin to grow.

cancerous and they form a lump, we call these lumps tumors.

Let me explain how a codon is altered:

*3.1.1. Virus*

are activated.

amino acids resulting in diseases.

We deciphered all 46 chromosomes. What surprises us most is that our genome contains 6,400,000,000 nucleotide bases, half from our father and half from our mother. Less than 2% of our genome contains genes which code for proteins. The other 98% of our genome contains switches, promoters, terminators, etc. The 46 chromosomes present in each cell of our body are the greatest library of the human book of life on planet Earth. The chromosomes carry genes which are written in nucleotides. Before sequencing (determining the number and the order of the four nucleotides on a chromosome), it is essential to know how many genes are present on each chromosome in our genome. The Human Genome Project has identified the following genes on each chromosome. We found that the chromosome (1) is the largest chromosome carrying 263 million A, T, G, and C nucleotide bases and has only 2610 genes. The chromosome (2) contains 255 million nucleotide bases and has only 1748 genes. The chromosome (3) contains 214 million nucleotide bases and carries 1381 genes. The chromosome (4) contains 203 million nucleotide bases and carries 1024 genes. The chromosome (5) contains 194 million nucleotide bases and carries 1190 genes. The chromosome (6) contains 183 million nucleotide bases and carries 1394 genes. The chromosome (7) contains 171 million nucleotide bases and carries 1378 genes. The chromosome (8) contains 155 million nucleotide bases and carries 927 genes. The chromosome (9) contains 145 million nucleotide bases and carries 1076 genes. The chromosome (10) contains 144 million nucleotide bases and carries 983 genes. The chromosome (11) contains 144 million nucleotide bases and carries 1692 genes. The chromosome (12) contains 143 million nucleotide bases and carries 1268 genes. The chromosome (13) contains 114 million nucleotide bases and carries 496 genes. The chromosome (14) contains 109 million nucleotide bases and carries 1173 genes. The chromosome (15) contains 106 million nucleotide bases and carries 906 genes. The chromosome (16) contains 98 million nucleotide bases and carries 1032 genes. The chromosome (17) contains 92 million nucleotide bases and carries 1394 genes. The chromosome (18) contains 85 million nucleotide bases and carries 400 genes. The chromosome (19) contains 67 million nucleotide bases and carries 1592 genes. The chromosome (20) contains 72 million nucleotide bases and carries 710 genes. The chromosome (21) contains 50 million nucleotide bases and carries 337 genes. Finally, the sex chromosome of all female called the (X) contains 164 million nucleotide bases and carries 1141 genes. The male sperm chromosome (Y) contains 59 million nucleotide bases and carries 255 genes.

If you add up all genes in the 23 pairs of chromosomes, they come up to 26,808 genes and yet we keep on mentioning 24,000 genes. The remaining genes are called the pseudogenes. For example, millions of years ago, humans and dog shared some of the same ancestral genes; we both carry the same olfactory genes. Since humans do not use these genes to smell for searching food, these genes are broken and they lose their functions in humans, but we still carry them. We call them pseudogenes. Recently, some Japanese scientists have activated the pseudogenes; this work may create ethical problem in future as more and more pseudogenes are activated.

The above DNA nucleotide bases constitute the genetic map of the normal human being; what makes them abnormal and makes us sick is the mutation in the coding regions of the genome. Now, we can examine the tumor genome of the oral cancer patients to identify specific mutations responsible for causing the disease. As I said above, less than 2% of the genome codes for amino acids. Slightest damage to the coding regions of the four nucleotides A, T, G, and C either by radiations, chemical pollution (from tobacco tar), genetic inheritance, or viral infection or by insertion, deletion, or inversion of the nucleotide bases code for wrong or abnormal amino acids resulting in diseases.

Although you and I are both human and yet no two individuals look the same because the AT/GC base pairs in each of us are arranged slightly differently, a difference of one nucleotide in a thousand base pair. The amazing fact is that out of 3,000,000,000 AT/GC base pairs, only 3 pairs of AT/GC code for a single amino acid, the building blocks of protein, called a codon (Nobel Prize was awarded to Nierenberg and Ochoa). Codons are the most important collections of AT/GC base pairs because they have correct instructions to make the right amino acids (there are only 20 amino acids; they randomly combine to make a protein). Thousands of amino acids make a protein and thousands of proteins make a cell; billions of cells make a tissue and hundreds of tissues make an organ and several organs make an individual such as you and me. This is how normal cell functions and we begin to grow.

As I said above, the old cells begin to die and they are constantly being replaced by healthy cells. Why do the normal cells become abnormal or become cancerous? Any factor that disrupts the 2% of the coding region of our genome will alter its function by slightly altering its code; an altered codon will code for a wrong amino acid and a wrong amino acid will give a wrong protein and it will make normal cell abnormal. When the functions of codons are disrupted intentionally or unintentionally, we alter the codon's function. For example, intentionally we alter a codon by smoking and unintentionally by exposure to environmental pollution such as chemicals or radiations. Altered codons have wrong information to make wrong amino acids. Wrong amino acids make wrong proteins and wrong proteins make wrong cells and wrong cells grow much faster than the normal cells and become abnormal or cancerous and they form a lump, we call these lumps tumors.

Four factors will disrupt a codon's function. Two are minor such as viruses and inherited oncogenes, and two are major such as radiations and chemical pollutants in our environment. Let me explain how a codon is altered:

#### *3.1.1. Virus*

gene is written in thousands of AT/GC base pairs that are linked together in a straight line and we call them DNA (deoxyribonucleic acid—Nobel prize was awarded to Crick and Watson for describing the double helical nature of the DNA structure). When thousands to millions of AT/GC base pairs contain information to make a single protein, we call that portion of AT/GC base pairs a gene (Nobel Prize was awarded to Khorana and Gilbert for making a functional gene). The genes begin to function with a start codon (AUG) and stop working at the following three stop codon: UGA, UGG, and UAG. After the stop codon, no more amino acids are added and DNA synthesis stops. If we count all the AT/GC base pairs in a single cell of our body, we will find that there are 3.2 billion pairs present in every cell. The entire AT/ GC sequence of 3.2 billion base pair is called the human genome or the book of our life which

We deciphered all 46 chromosomes. What surprises us most is that our genome contains 6,400,000,000 nucleotide bases, half from our father and half from our mother. Less than 2% of our genome contains genes which code for proteins. The other 98% of our genome contains switches, promoters, terminators, etc. The 46 chromosomes present in each cell of our body are the greatest library of the human book of life on planet Earth. The chromosomes carry genes which are written in nucleotides. Before sequencing (determining the number and the order of the four nucleotides on a chromosome), it is essential to know how many genes are present on each chromosome in our genome. The Human Genome Project has identified the following genes on each chromosome. We found that the chromosome (1) is the largest chromosome carrying 263 million A, T, G, and C nucleotide bases and has only 2610 genes. The chromosome (2) contains 255 million nucleotide bases and has only 1748 genes. The chromosome (3) contains 214 million nucleotide bases and carries 1381 genes. The chromosome (4) contains 203 million nucleotide bases and carries 1024 genes. The chromosome (5) contains 194 million nucleotide bases and carries 1190 genes. The chromosome (6) contains 183 million nucleotide bases and carries 1394 genes. The chromosome (7) contains 171 million nucleotide bases and carries 1378 genes. The chromosome (8) contains 155 million nucleotide bases and carries 927 genes. The chromosome (9) contains 145 million nucleotide bases and carries 1076 genes. The chromosome (10) contains 144 million nucleotide bases and carries 983 genes. The chromosome (11) contains 144 million nucleotide bases and carries 1692 genes. The chromosome (12) contains 143 million nucleotide bases and carries 1268 genes. The chromosome (13) contains 114 million nucleotide bases and carries 496 genes. The chromosome (14) contains 109 million nucleotide bases and carries 1173 genes. The chromosome (15) contains 106 million nucleotide bases and carries 906 genes. The chromosome (16) contains 98 million nucleotide bases and carries 1032 genes. The chromosome (17) contains 92 million nucleotide bases and carries 1394 genes. The chromosome (18) contains 85 million nucleotide bases and carries 400 genes. The chromosome (19) contains 67 million nucleotide bases and carries 1592 genes. The chromosome (20) contains 72 million nucleotide bases and carries 710 genes. The chromosome (21) contains 50 million nucleotide bases and carries 337 genes. Finally, the sex chromosome of all female called the (X) contains 164 million nucleotide bases and carries 1141 genes. The male sperm chromosome (Y) contains 59 mil-

If you add up all genes in the 23 pairs of chromosomes, they come up to 26,808 genes and yet we keep on mentioning 24,000 genes. The remaining genes are called the pseudogenes. For

carries total genetic information to make us.

38 Prevention, Detection and Management of Oral Cancer

lion nucleotide bases and carries 255 genes.

HPV causes more than 32,000 cases of cancer including oral cancer every year in the US. It is also very preventable by giving HPV vaccine for children at ages 11–12 which can protect them.

Viruses are not considered germ in the classical sense because they lack the ability to reproduce its progeny independently. To reproduce their own kind, viruses attack DNA of living cells, whether they are humans, animals, or plant cells. Viruses are also fragments of DNA that have the ability to merge in the host cell DNA and become integrated and invisible. They infect (merge) host's DNA and alter its functional machinery in such a way as to make protein and progeny for themselves. Not all viruses are bad. If all viruses kill their host cells, they will die too. Only a handful of virulent viruses destroy host cells. For example, AIDS viruses in human disrupt the codons and cause a unique cancer in AIDS patients called Kaposi carcinoma. Hardly anyone survives that cancer.

metastasized, the other organs lose their function; with too many defected organs, a patient cannot survive for long. When we examine the internal structure of tumors of a dead patient,

The Impact of Sequencing Human Genome on Drug Design to Treat Oral Cancer

http://dx.doi.org/10.5772/intechopen.80231

41

Let me tell you what you can do to protect yourselves and what we can do to help you? If you want to protect yourselves from oral or lung cancers, stop taking tobacco in any form and in any kind. The best way we can help you is to pursue as vigorously as we could do to find other sensitive genes and try to replace them with healthy genes by a method called gene therapy. Gene therapy could work if a single gene is mutated. Unfortunately, several genes are mutated in oral lung cancers. Gene therapy fails, but drug therapy works. From here my

As I said above, although the book of life is written in 3.2 billion AT/GC base pairs, about 2% of the AT/GC base pairs contain 24,000 genes (specific instructions to make proteins), the rest of DNA is called the junk DNA. As I said above, it is not garbage that we throw away; it contains important gene switches, promoters, enhancers, etc. We keep it because someday we might be able to find out what additional information it may provide about us. We carry all 24,000 genes in every cell of our body, but less than 6000 genes are probably bad genes (mutated) that are linked to a variety of genetic defects leading to 6000 different diseases. Each of us does not carry all 6000 bad genes, but we do carry a single copy of at least 4–5 bad genes in our genome. They remain dormant because only one parent carries a single copy of a mutated gene. We need both bad copies of bad genes, one from each parent to get sick. We have inherited these bad genes from our parents. Our parents inherited these genes from their parents. One ancestor can pass on the same bad genes to her children and children's children. Therefore, it is a wise idea not to marry within the same family tree. The more intermarriages among the same family members, the more bad genes tend to concentrate among fewer and fewer children of that family.

I could take tobacco extract samples to the lab and analyze its ingredients. First, I soak the tobacco in ammonia and extract with chloroform. All nitrogen bases are extracted in chloroform. I could wash the extract with water to remove all water-soluble impurities and dry the extract and distill off the chloroform. I place the residue in long glass column filled with silica gel. I pour a solvent mixture which carries the residue down the column. It is called the separation of different components by chromatography. I shine the UV light on the glass column as the solvent flows down. Hundreds of different bands appear in different colors, corresponding to hundreds of components present in the tobacco tar. The largest band is nicotine. To confirm, I take a little sample from the band and inject in the MS (mass spectrometer: the largest peak corresponds to the molecular weight of the nicotine). I could make the radiolabeled nicotine by adding C-14 diazomethane in sodium hydroxide. The reaction adds a radiolabeled methyl group to nicotine molecule. I inject the radiolabeled methyl nicotine to half a dozen mice. I collect their urine samples separately. Analysis of the urine sample shows that some mice produce no change and others produce a new chemical called nicotine N-oxide. All those mice in which the gene that produces monoamine oxidase was activated developed cancer. If you inject nicotine N-oxide to another set of a dozen mice, they all come

down with various cancers. All aromatic N-oxides are carcinogens.

work begins. Professor Ross and I design drugs to shut off multiple defected genes.

we find it mostly consists of abnormal cells that have been altered.

*3.1.3. Nicotine N-oxide is a carcinogen*

#### *3.1.2. Oncogene*

Oncogenes are also fragments of mutated DNA, but they are always bad. They are complete genes that have the instructions to make a specific bad protein. Such genes are called oncogenes (cancer-causing genes). Our institute's, NIH's, past Director, Dr. Harold Varmus, was the first man to identify an oncogene in humans. For his work, Dr. Varmus shared a Nobel Prize with Dr. Michael Bishop.

From here begins one of the most exciting stories that explain the causes of cancer. In the early 1990s, some scientists in England were studying cancer-causing viruses. When scientists injected cancer-causing viruses to animals, they found that sometimes viruses grow and other times they do not. On close examination, they detected a background protein in all cells. Whenever the background protein is absent, cancer-causing viruses grow and the animal develops cancer. Whenever the background protein is present, cancer-causing viruses will not grow. They called it the background Protein 53 or (P53). They identify the gene that makes P53 protein and they named P53 gene. Since cancer is suppressed in the presence of P53, scientists named P53 as cancer suppressor proteins. When a normal cell is damaged, the surrounding cells grow to make P53 protein to repair the damage. When healing is complete, the P53 protein stops the cells from further growth. If there is a mutation in the P53 gene, P53 loses its function and the cell growth will not stop and grow continuously and become cancerous.

As I said above, that gene is a collection of codons and each codon is made of three pairs of AT/ GC. Hundreds of thousands to millions of AT/GC base pairs combine to form gene-53. Now, we know that the codon in P53 is sensitive to mutation by chemicals, radiations, viruses, and oncogenes. If you cause a slight defect in the codon by altering one letter of AT/GC base pair, the entire P53 gene becomes defected. Defected P53 cannot produce background protein that suppresses cancer. In the absence of P53 protein, patients develop cancer. The lesson we learn is that if a single letter of this four-letter AT/GC base pairs is altered by virus or by chemicals or by gene or by radiation, first a single normal cell becomes abnormal or cancerous. Over many replications, the mutated cell becomes cancerous. Repeated exposure to chemicals such as smoking tobacco several times a day, you could alter a single letter of AT/GC base pairs. If a single cell becomes defected, it will multiply and accumulate and an entire organ becomes cancerous. When the defected organ cannot function, we become ill.

Unfortunately, cancer is not localized at one place for long (we could have cut out the defected organ and throw it away); it spreads or metastasizes as I described above. Once it is metastasized, the other organs lose their function; with too many defected organs, a patient cannot survive for long. When we examine the internal structure of tumors of a dead patient, we find it mostly consists of abnormal cells that have been altered.

Let me tell you what you can do to protect yourselves and what we can do to help you? If you want to protect yourselves from oral or lung cancers, stop taking tobacco in any form and in any kind. The best way we can help you is to pursue as vigorously as we could do to find other sensitive genes and try to replace them with healthy genes by a method called gene therapy. Gene therapy could work if a single gene is mutated. Unfortunately, several genes are mutated in oral lung cancers. Gene therapy fails, but drug therapy works. From here my work begins. Professor Ross and I design drugs to shut off multiple defected genes.

As I said above, although the book of life is written in 3.2 billion AT/GC base pairs, about 2% of the AT/GC base pairs contain 24,000 genes (specific instructions to make proteins), the rest of DNA is called the junk DNA. As I said above, it is not garbage that we throw away; it contains important gene switches, promoters, enhancers, etc. We keep it because someday we might be able to find out what additional information it may provide about us. We carry all 24,000 genes in every cell of our body, but less than 6000 genes are probably bad genes (mutated) that are linked to a variety of genetic defects leading to 6000 different diseases. Each of us does not carry all 6000 bad genes, but we do carry a single copy of at least 4–5 bad genes in our genome. They remain dormant because only one parent carries a single copy of a mutated gene. We need both bad copies of bad genes, one from each parent to get sick. We have inherited these bad genes from our parents. Our parents inherited these genes from their parents. One ancestor can pass on the same bad genes to her children and children's children. Therefore, it is a wise idea not to marry within the same family tree. The more intermarriages among the same family members, the more bad genes tend to concentrate among fewer and fewer children of that family.

#### *3.1.3. Nicotine N-oxide is a carcinogen*

Viruses are not considered germ in the classical sense because they lack the ability to reproduce its progeny independently. To reproduce their own kind, viruses attack DNA of living cells, whether they are humans, animals, or plant cells. Viruses are also fragments of DNA that have the ability to merge in the host cell DNA and become integrated and invisible. They infect (merge) host's DNA and alter its functional machinery in such a way as to make protein and progeny for themselves. Not all viruses are bad. If all viruses kill their host cells, they will die too. Only a handful of virulent viruses destroy host cells. For example, AIDS viruses in human disrupt the codons and cause a unique cancer in AIDS patients called Kaposi carci-

Oncogenes are also fragments of mutated DNA, but they are always bad. They are complete genes that have the instructions to make a specific bad protein. Such genes are called oncogenes (cancer-causing genes). Our institute's, NIH's, past Director, Dr. Harold Varmus, was the first man to identify an oncogene in humans. For his work, Dr. Varmus shared a Nobel

From here begins one of the most exciting stories that explain the causes of cancer. In the early 1990s, some scientists in England were studying cancer-causing viruses. When scientists injected cancer-causing viruses to animals, they found that sometimes viruses grow and other times they do not. On close examination, they detected a background protein in all cells. Whenever the background protein is absent, cancer-causing viruses grow and the animal develops cancer. Whenever the background protein is present, cancer-causing viruses will not grow. They called it the background Protein 53 or (P53). They identify the gene that makes P53 protein and they named P53 gene. Since cancer is suppressed in the presence of P53, scientists named P53 as cancer suppressor proteins. When a normal cell is damaged, the surrounding cells grow to make P53 protein to repair the damage. When healing is complete, the P53 protein stops the cells from further growth. If there is a mutation in the P53 gene, P53 loses its function and the cell growth will not stop and grow continuously and become cancerous.

As I said above, that gene is a collection of codons and each codon is made of three pairs of AT/ GC. Hundreds of thousands to millions of AT/GC base pairs combine to form gene-53. Now, we know that the codon in P53 is sensitive to mutation by chemicals, radiations, viruses, and oncogenes. If you cause a slight defect in the codon by altering one letter of AT/GC base pair, the entire P53 gene becomes defected. Defected P53 cannot produce background protein that suppresses cancer. In the absence of P53 protein, patients develop cancer. The lesson we learn is that if a single letter of this four-letter AT/GC base pairs is altered by virus or by chemicals or by gene or by radiation, first a single normal cell becomes abnormal or cancerous. Over many replications, the mutated cell becomes cancerous. Repeated exposure to chemicals such as smoking tobacco several times a day, you could alter a single letter of AT/GC base pairs. If a single cell becomes defected, it will multiply and accumulate and an entire organ becomes

Unfortunately, cancer is not localized at one place for long (we could have cut out the defected organ and throw it away); it spreads or metastasizes as I described above. Once it is

cancerous. When the defected organ cannot function, we become ill.

noma. Hardly anyone survives that cancer.

40 Prevention, Detection and Management of Oral Cancer

Prize with Dr. Michael Bishop.

*3.1.2. Oncogene*

I could take tobacco extract samples to the lab and analyze its ingredients. First, I soak the tobacco in ammonia and extract with chloroform. All nitrogen bases are extracted in chloroform. I could wash the extract with water to remove all water-soluble impurities and dry the extract and distill off the chloroform. I place the residue in long glass column filled with silica gel. I pour a solvent mixture which carries the residue down the column. It is called the separation of different components by chromatography. I shine the UV light on the glass column as the solvent flows down. Hundreds of different bands appear in different colors, corresponding to hundreds of components present in the tobacco tar. The largest band is nicotine. To confirm, I take a little sample from the band and inject in the MS (mass spectrometer: the largest peak corresponds to the molecular weight of the nicotine). I could make the radiolabeled nicotine by adding C-14 diazomethane in sodium hydroxide. The reaction adds a radiolabeled methyl group to nicotine molecule. I inject the radiolabeled methyl nicotine to half a dozen mice. I collect their urine samples separately. Analysis of the urine sample shows that some mice produce no change and others produce a new chemical called nicotine N-oxide. All those mice in which the gene that produces monoamine oxidase was activated developed cancer. If you inject nicotine N-oxide to another set of a dozen mice, they all come down with various cancers. All aromatic N-oxides are carcinogens.

developing drugs on a rational basis and for treating various cancers including the oral cancer. The technologies developed during the completion of the Human Genome Project (the tool kits which contain hundreds of restrictions enzymes to cut DNA ligase to join DNA pieces; using these tool kits, we can cut, paste, and copy genes) could be used to treat and prevent oral cancer. The recently completed Thousand Genome Project will pinpoint with precision and accuracy the specific damage to DNA nucleotides responsible for causing various oral cancers. Once the mutated genes on a specific chromosome are identified, we can design drugs to shut off those genes like we designed AZQ (US Patent 4,146,622) to attack brain tumor (for

The Impact of Sequencing Human Genome on Drug Design to Treat Oral Cancer

http://dx.doi.org/10.5772/intechopen.80231

43

Once the mutation sites and chromosome numbers are identified, we can diagnose, prevent, and treat the oral cancer either by gene therapy if a single gene mutation is responsible for causing any of the above cancers or by drug therapy if multiple mutations are involved. As I stated above, French Anderson and his colleagues have successfully developed gene therapy for treating SCID (severe combined immunodeficiency) syndrome; we could use the same method to cut and paste and replace the bad gene with the good gene in a virus which is used to infect the WBC obtained from the same patient. After harvesting the infected WBC, the transgenic WBC was injected back in the same patient to treat SCID. It worked and patients fully recovered. Several thousand SCID children are living a normal life. Gene therapy works with a single gene mutation, but not if the multiple mutations are responsible

On the other hand, if cancer is caused by multiple mutations, we could use drug therapy by preventing malignant cell replication developed by Ross by cross-linking both strands of DNA. Using dyes specific to OC cells as carriers for nitrogen mustard, as done by Ross in making Melphalan, we could also develop new class of drugs to attack cancer cells in the other parts of the oral cavity. The bad news is that there have been 13 different forms of oral cancer identified. The good news is that for designing a drug, we have to find a dye which colors one of these tumors. There are hundreds of dyes available for testing. Once we succeed in finding a dye, we could design drugs by using our method by attaching aziridines to attack that specific oral cancer by shutting off mutated genes by binding to a single strand of DNA. What would happen if we succeed and when next-generation sequencers produce inexpensive and fast sequencing genomes is that we could identify all mutated genes on all 13 oral cancers with precision and accuracy and design drugs to shut off those genes.

In the laboratory of the Sir Walter Ross at the Royal Cancer Hospital of London University, England, I was trained to design drugs to attack mutated DNA shutting off mutated genes. Professor Ross had spent all his life working on "Biological Alkylating Agents" and published a series of paper including a book [9–13]. Using the same rationale, I worked with Professor Ross for almost 10 years at London University developing anticancer drugs. Instead of crosslinking DNA with nitrogen mustards, I used aziridines to bind to a single strand of DNA

structure, see **Figure 1**).

for causing diseases.

shutting off the genes.

**4. Historical background for drug design**

**Figure 1.** NIH Scientific Achievement Award. Aziridines as single strand DNA binding agent.

Now, you know why Sir Winston Churchill, who smoked cigar all his life, never developed cancer, but film actor Yul Brenner smoked cigarettes and died of lung cancer. If you were to analyze their urine samples, you will find that Mr. Brenner urine contains nicotine N-oxide. The gene monoamine oxidase is activated in Brenner to make nicotine N-oxide not in Sir Winston. While Sir Winston lived, Brenner died.

While I was busy designing drugs, such as AZQ, to shut off genes which cause brain cancers, my colleagues in the other labs at NCI (National Cancer Institute) have isolated hundreds of chemicals from the tobacco tar which contains dozens of carcinogenic chemicals. If you would apply the tobacco tar on the skin of mice, within a few weeks, tumor develops on the skin surface. The major culprit is nicotine which is considered as one of the most addictive chemicals. Some studies showed that it is even more addictive than many known narcotics such as marijuana, opiates, and heroin. Oral cancer (OC) is caused by chemicals released by chewing tobacco. Most football players chew tobacco; they call it smokeless tobacco. Smoking burns tobacco generating even more aromatic amines which are known carcinogens. Nitrosoamines bind to DNA producing mutations.

After the completion of the Human Genome Project, we have identified specific mutations responsible for a specific disease. Now, we design drugs to attack that specific mutation to shut off that gene. The completion of the Human Genome Project has the greatest impact on developing drugs on a rational basis and for treating various cancers including the oral cancer. The technologies developed during the completion of the Human Genome Project (the tool kits which contain hundreds of restrictions enzymes to cut DNA ligase to join DNA pieces; using these tool kits, we can cut, paste, and copy genes) could be used to treat and prevent oral cancer. The recently completed Thousand Genome Project will pinpoint with precision and accuracy the specific damage to DNA nucleotides responsible for causing various oral cancers. Once the mutated genes on a specific chromosome are identified, we can design drugs to shut off those genes like we designed AZQ (US Patent 4,146,622) to attack brain tumor (for structure, see **Figure 1**).

Once the mutation sites and chromosome numbers are identified, we can diagnose, prevent, and treat the oral cancer either by gene therapy if a single gene mutation is responsible for causing any of the above cancers or by drug therapy if multiple mutations are involved. As I stated above, French Anderson and his colleagues have successfully developed gene therapy for treating SCID (severe combined immunodeficiency) syndrome; we could use the same method to cut and paste and replace the bad gene with the good gene in a virus which is used to infect the WBC obtained from the same patient. After harvesting the infected WBC, the transgenic WBC was injected back in the same patient to treat SCID. It worked and patients fully recovered. Several thousand SCID children are living a normal life. Gene therapy works with a single gene mutation, but not if the multiple mutations are responsible for causing diseases.
