Tumor marker : Introduction, Classification and Clinical application

Introduction:

The first tumor marker reported was the Bence-Jones protein (monoclonal light chains of Ig secreted by tumor plasma cells) by precipitation in acidified boiled urine and is the diagnostic marker of multiple myeloma. It was identified by H. Bence-Jones in 1846. The second era (1982-1963) was the discovery of hormones, enzymes, isoenzymes and proteins

How to study Biochemistry ?

Biochemistry is often cited as a challenging, high-volume course that revolves around complex molecular relationships and multi-step reactions.  Successful students must have a strong understanding of the concepts and solid memorization techniques to succeed in this course.  The following study strategies and tips have been formulated from past students that succeeded in their courses to help you learn how to study Biochemistry.

Study Skill #1 - Do NOT procrastinate.  The most obvious, and yet least followed advice by students.  Biochemistry is a high-volume course that progresses and builds its concepts on the fundamentals.  Moreover, many pathways and reactions require memorization and must be acquired over time.

Study Skill #2 - Start with the big picture.  There is no doubt that you will have to memorize multi-step metabolic pathways.  The best way to do this is to start with the big steps and understand the overall flow of the reaction.  First, write only the substrates and products in order.  Do this repeatedly, until it is memorized.  Then add the enzymes.  Then continue to  add co-factors and by-products.  If necessary, label each as an exer- or endergonic reaction and whether or not it is reversible.  Check out Lippincott's Illustrated Reviews: Biochemistry Book for an easy-to-understand approach and large full-color illustrations.  Use the nomenclature to help you remember what is going on in each step. For example, Phosphofructokinase-1, adds a phosphate group (phospho-kinase) to the molecule fructose (-fructo-) at the first position (-1).  By breaking down the pathways and focusing on the terminology it will greatly speed up your ability to memorize them.

Study Skill #3 - Know the terminology and nomenclature, it will make things much easier down the road.  An enzyme or protein will often have its function built into its name.  Take Protein Kinase A for example.  As a member of the Kinases, it will almost always add a phosphate group to its substrate.  Or, take Alcohol Dehydrogenase, structures that are Dehydrogenases always oxidize a substrate.  In this case, it oxidizes alcohols into aldehydes and ketones.  Once you get this down, you will begin to recognize names and automatically correlate them with a specific function.

Study Skill #4 - Buy a dry erase board.  Use this to memorize the pathways and any other reactions you have to know.  There are no short-cuts, but writing things out reinforces them in your memory.  It tends to be much more efficient than staring and reciting from your textbook.

Study Skill #5 - Know the purpose of a reaction. Take the Bohr Effect for example.  An increase in [H+] (decrease in pH), CO2, temperature, and 2,3-BPG all occur in active skeletal muscle. They also all encourage O2 release from hemoglobin.  This makes sense if you think that working muscle is metabolic tissue and needs oxygen to survive.  Incorporating the larger concept will also allow you to predict the flow of reactions in other situations throughout the body. 

Study Skill #6 - Stare at the graphs and plots.  These questions are virtually freebies on exams because all the information you need to solve them is included.  Know what the x- and y-intercept, the slope, and the area under the graph represent.  Know what makes the graphed line move to the right or left.  You will absolutely be asked about the Michaelis-Menten graph and the Hemoglobin dissociation curve - these are staples of biochemistry.

Study Skill #7 - Seek to understand first, and then memorize.  Like many other courses, biochemistry can be overwhelming at first.  There is no easy way to memorize every amino acid or metabolic reaction.  But students always claim that if they take the time to first get the concept down, the memorizing is not as difficult as it once seemed.  Stay focused, break it down into small steps, and practice.

(Source: www.medstudysites.com)

Human Genome Project: Human Genome and Human Gene Therapy

The Human Genome and Human Gene Therapy:

The strategy of human gene therapy is the introduction of DNA into human cells to correct inherited genetic deficiencies.

E.g. Severe Combined Immune Deficiency (SCID). One form of SCID results from genetically inherited defects in gene encoding adenosine Deaminase (ADA), an enzyme involved in nucleotide biosynthesis. Another form of SCID arises from defect in cell surface receptor for Interleukin which triggers differentiation. In both case the progenitor stem cells cannot differentiate into mature immune cells like T and B lymphocytes. Children with this disorder are highly susceptible to bacterial and viral infections.

A gene therapy trial initiated in 1999 was successful in correcting a form of SCID caused by defective cytokine receptors. The researchers introduced the corrected gene for γc cytokine receptor subunit in retrovirus and this was again transfected into CD34+ cells (the stem cells that give rise to immune system cells). The transformed cells were placed back into  the patient’s bone marrow. The corrected gene conferred a growth over untreated cells. A functioning immune system was detected. Although two of patients developed severe form of leukemia due to retrovirus itself got integrated itself into a chromosome of stem cells resulting in abnormally high expression of hematopoietic oncogene that led to uncontrolled cell growth.

The first human gene therapy trial was carried out at National Institute of Health in Bethesda, Maryland in 1990. The patient was a four year old girl crippled by ADA deficiency. Bone marrow cells from the child were transformed with an engineered retrovirus containing a functional, ADA gene. The transformed cells in vivo were introduced into the patient’s marrow. Four years later, the child was leading a normal life.

Although the  gene  therapy is absolute  care it is associated with various risks. One major impediment proved to be the inefficiency of introducing new genes into cells. Transformation failed in many cells, and the number of transformed cells often proved insufficient to reverse the disorder.

Human gene therapy is not limited to genetic diseases. Cancer cells are being targeted by delivering genes for proteins that might destroy the cell or restore the normal cell division. Immune system cells associated with tumors can be genetically modified to produce tumor necrosis factor (TNF). When these lymphocytes are taken from a cancer patient, modified, and reintroduced, the engineered cells target the tumor, and the TNF they produce causes tumor shrinkage. AIDS may also be treatable with gene therapy; DNA that encodes an RNA molecule complementary to a viral HIV mRNA could be introduced into immune system cells (the targets of HIV). The RNA transcribed from the introduced DNA would pair with the HIV mRNA, preventing its translation and interfering with the virus's life cycle.

Human Genome Project:

Discussion in mid 1980 led to initiation of the Human Genome Project in 1989 but the actual work of sequencing began at 1990. Researchers team first generated a detailed physical map of human genome with clones derived from each chromosome.

Fig: The Human genome project strategy: Sequenced by shotgun sequencing.

Human genome project was completed in 2001 and published in April 2003. We have only 25, 000 – 30, 000 genes and 3.9 X 109 bp per haploid genome i.e. 23 chromosomes. 1.1 to 1.4% of our DNA actually encodes proteins. More than 50% of our genome consist of short repeated sequences the vast majority of which about 45% come from transposons. The human genome

can encode 30,000 to 40,000 proteins. There are more than 3 million SNPs. Whole genome contains 10% of Alu elements and present in GC rich region. This project was handled by international collaboration involving groups from the USA, UK, Japan, France, Germany and china known as International Human Genome Sequencing Consortium (IHGSC).

The human and chimpanzee genome differ by only 1.2% at the level of base pairs and even less in gene encoding proteins. This creates 35 million base pair changes.

The overall approach, referred to as hierarchical shotgun sequencing, consisted of fragmenting the entire genome into pieces of approximately 100–200 kb and inserting them into bacterial artificial chromosomes (BACs). The BACs were then positioned on individual chromosomes by looking for marker sequences known as sequence-tagged sites STSs), whose locations had been already determined. STSs are short (usually < 500 bp), unique genomic loci for which a PCR assay is available. Clones of the BACs were then broken into small fragments (shotgunning). Each fragment was then sequenced,  and computer algorithms were used that recognized matching sequence information from overlapping fragments to piece together the complete sequence.

On the basis of STS, PCR based sequencing was done using primers that recognize the vector terminal sequence and using labeled dNTPs. ddNTP was used to terminate the chain elongation process to generate short fragments formed due to addition of ddNTP in the complementary site. These fragments were then sequences and assembled to determine the overlapping bases on the basis of these overlap fragments from entire genome fragments were joined to produce final sequence.

For More information on Human Genome Project, Visit:

http://www.genome.gov/10001772

(Source: Lehninger's Textbook of Biochemistry)

Transgenic Animals and DNA Micro-array

Transgenic animals:

The strategy here is injection of genes in fertilized mouse ovum, these genes will be incorporated into the genome and found in both somatic and germ cells. By this strategy many transgenic animals have been established and are studied for tissue specific effect on gene expression and effects of overproduction of gene products. The transgenic approach has been used to correct a genetic deficiency in mice. Fertilized ova obtained from mice with genetic hypogonadism were injected with DNA containing the coding sequence for the gonadotropin- releasing hormone (GnRH) precursor protein. This gene was expressed and regulated normally in the hypothalamus of certain number of mice, and these animals were in all respects normal.

Application of Restriction Fragment Length Polymorphism (RFLP)

DNA-based screening is useful not only in determining if an unborn fetus is affected, but also in detecting carriers of the mutated gene. PKU, like many inborn errors of amino acid metabolism, is inherited as an autosomal recessive trait. Identification of heterozygotes can aid in future family planning.

Another approach of detecting the βs globin mutation is by the use of Allele specific oligonucleotide probe (ASO). If PCR is carried out using such primer then it is called ASO-PCR.