M.Prasad NaiduMSc Medical Biochemistry,

Ph.D.Research Scholar

Introduction Cancer cells are characterized by three properties :

1.Diminished or unrestrained control of growth ,2.Invasion of local tissues ,3.Spread or metastasis to other parts of the

body .• Nearly all cancers originate from a single cell

this clonal origin is critical discriminating feature between neoplasia & hyperplasia .

contdPhysical , chemical , & biological agents can

cause cancer .Agents causing cancer fall into 3 broad

groups :1.Radiant energy ,2.Chemical compound ,3.Viruses .Spontaneous mutations Oxidative damage to DNA .

Carcinogenic chemicals

Both organic & inorganic molecules are carcinogenic . Carcinogens do not share structural similarity .

Ultimate carcinogens are electrophiles ( molecules deficient in electrons ) they readily attack nucleophilic groups ( electron rich ) in DNA , RNA , & proteins .Metabolism of procarcinogens involves cytochrome P 450 system .

Procarcinogen

Proximate carcinogen

Ultimate carcinogen

contdCarcinogens interact covalently with cellular

macro molecules .

Carcinogens found to interact with purine , pyrimidine , or phosphodiester groups of DNA .

The most common site of attack is guanine & addition of various carcinogens to N2 , N3 , N7 , O6 , O8 atoms of this base .

contdPersistant unrepaired lesions are important in

generating mutations critical for carcinogenesis.

Ames assay is used to detect the mutagenecity of the chemical carcinogen .

Ames assay uses specially constructed strain of salmonella typhimurium , that has a mutation in gene that codes for one of the enzyme involved in synthesis of histidine .

S .typhimurium of the above strain can not synthesize histidine , needs histdine added to the medium for it’s growth .

Chemical carcinogen by its mutagenic nature restore synthesis of histidine in the S . Typhimurium .

S .typhimurium along with mitochondrial supernatant provides activation by monooxygenases .

Initiation & promotion • In organs such as skin & liver carcinogenesis

has 2 stages

1.Intiation ( rapid & irreversible stage ),2.Promotion .

• Most carcinogens are capable of acting as both intiating & promoting agents .

DNA the critical macromolecule in carcinogenesis Cancer cells beget cancer cells that is

essential changes responsible for cancer are transmitted from mother to daughter cells .

Both irradiation & chemical carcinogens damage DNA & are capable of causing mutations in DNA .

Many tumor cells exhibit abnormal chromosomes .

Transfection experiments indicate that purified DNA ( oncogens ) from cancer cells can transform normal cells into ( potential )cancer cells .

Genes that increase susceptibility to cancer have been isolated .

Epigenetics is defined as changes that alter the pattern of gene expression that persists across at least one cell division eg: methylation of CpG dinucleotides , histone acetylation .

Oncogenes play a crucial role in carcinogenesisAn oncogene is a gene that, when mutated or

expressed at high levels, turn a normal cell into a tumor cell.

Oncogenes were first recognized as unique genes of tumor causing viruses that are responsible for the process of transformation.

Oncogenes of Rous Sarcoma virusRous sarcoma virus is retrovirus containing 4

genes named gag , pol , env , src .

contd gag gene encodes for group specific antigens of

the virus .

pol for reverse transcriptase that characterizes retro viruses .

env for certain glycoproteins of viruses .

src ( sarcoma causing gene ) produces a protein tyrosine kinase .

contd

The abnormal phosphorylation of vinculin a protein in focal adhesion plaques could help explain the rounding - up of cells & their diminished adhesion to substratum & to one another during transfromation .

Certain glycolytic enzymes appear to be target proteins for src tyrosine kinases .

contd

The products of src interact with the kinase catalyzing phosphorylation of phosphotidyl inositol to phosphotidyl inositol 4 , 5 bis phosphate .

Inositol triphosphate releases calcium from intracellular storage .

Contd Diacyl glycerol activates protien kinase C

which inturn phosphorylates number of proteins some of which are ion pumps .

Mild alkalinization of the cell brought about by activation Na+ / H+ antiport system could play a role in stimulating mitosis .

Protein tyrosine kinase in normal & transformed cellsInsulin , PDGF , EGF found in both normal &

transformed cells have tyrosine kinase activity .

The amount of phosphotyrosine in most normal cells is low but is usually elevated in cells transformed by oncogenic virus containing protein tyrosine kinase .

contdThe product of ras oncogene of murine

sarcoma virus binds GTP , has GTPase activity & regulate activity of adenylyl cyclase .

myc oncogen encodes a DNA binding protein .

abl , src , sis , erb – B , oncogene products have tyrosine kinase activity .

Proto - Oncogene A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression .

Products of proto – oncogenes believed to play important roles in normal differentiation & other cellular process.

Oncogenes from tumor cells DNA isolated from tumor cell

addedRecipient cells often a line of mouse fibroblasts

NIH/3T3 microscopic observation for1-2 wk

Foci of transformed cell

contd

The procedure is repeated several times using DNA extracted from transformed cells ,thus reducing the amount of DNA not involved in transformation , that was transfected & facilitating identification by southern blot technique using suitable probe .

Activation of Proto - oncogenes 5 mechanisms of activation :1. Promoter insertion ,2. Enhancer insertion ,3. Chromosomal translocation ,4. Gene amplification ,5. Point mutations .

Promoter Insertion Certain retro viruses lack oncogenes ( eg :

avian leukemia viruses ) but may cause cancer over a long period of time .

Retroviruses infect cells a DNA copy ( cDNA ) of their RNA genome is synthesized by reverse transcriptase & the cDNA is integrated into the host genome .

Enhancer Insertion

Chromosomal translocation

Mechanism of action of oncogens

Oncogens act on key intracellualr pathways involved on growth control , uncoupling them from the need fro an exogenous stimuli .

Products of src acting as tyrosine kinase , products of ras acting to stimulate adenylyl cyclase act by phosphorylating key regulatory proteins of cell cycle .

Elevations of various cyclins , decrease of kinase inhibitory proteins ( KIP : p21 ,p27, p57 ) & decrease of inhibitors of CDK4 ( INK4 : p16 )has been documented in cancers .

Growth factors Polypeptide growth factors are mitogenic .

Growth factors act in an endocrine , paracrine , or autocrine manner .

Growth factors act on the cell cycle & mitosis via transmembrane signal transduction.

Growth factors & oncogenes interact in several ways The products of several oncogenes are either

growth factors or parts of the receptors for growth factors .

Product of sis oncogene truncated B chain of PDGF .

B chain is biologically active as homodimer without involvement of A chain .

contdAutocrine stimulation by PDGF gives a chronic

mitogenic stimulus could be an imporatnt factor in transformation of cell .

The product of erb – B is truncated EGF with much of deletion of external domain of EGF but retaining the tyrosine kinase part .

This abnormal form is continuously active tyrosine kinase when present in cells causing chronic mitogenic stimulus .

Transforming growth factor βTGFβ known to inhibit the growth of most cell

types except fibrobalsts .

In fibroblast TGFβ promotes growth by activating sis gene .

TGFβ inhibitory effect is on cell cycle progression is by phosphorylating pRB , reduction of the levels of mRNA s of cyclin E & A , inhibition of cyclin E & cyclin A dependent kinases .

The p53 tumor suppressor gene acts as a guardian of the genome

Mutations in p53 occurs frequently in many human tumors .

Selenium has cancer preventive action .

Selenomethionine the main form of selenium in our diets , participates in a redox reaction resulting in the reduction of 2 cysteine residues within p53 leading to an induction of p53 DNA – binding activity .

HPV proteins E6 & E7 bind & inactivate cellular tumor suppressors p53 & pRB respectively .

Genetic model of colorectal cancer suggest poly gene etiology for its development Multiple cumulative mutational events are

invariably required for the progression from normal to fully malignant phenotype .

Telomerase DNA polymerase is unable to replicate the ends of

chromosomes , resulting in loss of DNA at specialized ends of chromosomes called telomere.

Telomeres composed of tandem repeates of six nucleotide sequences ( TTAGGG ) .

contdTelomere binds with specialized telomere

binding proteins to form a T loop structure that prevents the ends of chromosomes from being recognized as broken or damaged DNA.

Loss of telomere repeats with each cell division cycle causes gradual telomere shortening leading to growth arrest.

contd

Critically short telomere triggers a p53 regulated DNA damage check point , this is called replicative senescence .

Cells can bypass this growth arrest if pRB or p53 are nonfunctional .

contdThe ability to bypass telomere based growth

limitations is thought to be a critical step in the evolution of most malignancies .

This occurs by the expansion of telomerase in cancer cells .

Telomerase is an enzyme that adds TTAGGG repeats onto the 3’ end of chromosome .

Contd More than 90% of human cancers express

high levels of telomearse .

Most normal do not express sufficient telomerase to prevent telomere attrition with each cell division .

Stems cell have high telomerase activity .

Malignancy of tumor cells tends to progressOnce a cell becomes tumor cell , the composition & behavior of it’s progeny do not remain static , there is tendency for malignancy to increase .

The important phenomenon of progression appears to reflect a fundamental instability of the genome of tumor cells .

Metastasis is the most dangerous property of tumor cells Metastasis is spread of cancer cell from primary site of origin to other tissue where they grow as secondary tumors .

There is failure of cell - cell interaction much attention is on comparison of the biochemistry of normal & malignant cells .