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Lecture Notes

Methotrexate

Methotrexate is an antimetabolite. A broad definition of antimetabolites would include compounds with structural similarity to precursors of purines or pyrimidines, or compounds that interfere with purine or pyrimidine synthesis.

Antimetabolites can cause DNA damage indirectly,

  • through misincorporation into DNA,
  • abnormal timing or progression through DNA synthesis,
  • or altered function of pyrimidine and purine biosynthetic enzymes.

 They tend to convey greatest toxicity to cells in S-phase (figure-1) and the degree of toxicity increases with duration of exposure.

 Action of methotrexate

Figure-1- Cell cycle summary and the action of methotrexate that acts in the S phase of the cell cycle

Common toxic manifestations include

  • stomatitis,
  • diarrhea, and
  • myelosuppression.

Mechanism of Action

Methotrexate inhibits Dihydro folate reductase, which regenerates reduced folates from the oxidized folates (figure-2 and 4)  produced when thymidine monophosphate is formed from deoxy uridine monophosphate (Figure-4) Without reduced folates, cells die a “thymine-less” death.

 Conversion of dihydro folate to tetra hydro folate

Figure-2- Methotrexate is a competitive inhibitor of Dihydrofolate reductase

Overview of role of folic acid in DNA synthesis

Folate is an essential vitamin, found in green leafy vegetables. It is essential for many biochemical processes in the body, including DNA synthesis and red blood cell synthesis

Tetrahydrofolate (THF) derived from the vitamin folic acid is the major source of 1-carbon units, used in the biosynthesis of many important biological molecules. This cofactor is a carrier of activated 1-carbon units at various oxidation levels (methyl, Methylene, Formyl, Formimino, and methenyl). These compounds can be interconverted as required by the cellular process. (See figure-3)

 Forms of tetrahydro folate

 Figure-3- forms of Tetrahydrofolate (THF).

Role of folic acid in the synthesis of pyrimidine nucleotides

All cells, especially rapidly growing cells, must synthesize Thymidylate (dTMP) for DNA synthesis. The difference between (T) and (U) is one methyl group at the carbon-5 position. Thymidylate is synthesized by the Methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme Thymidylate synthase. This reaction requires a methyl donor and a source of reducing equivalents, which are both provided by N5, N10-methylene THF)-figure-3 and 4. For this reaction to continue, the regeneration of THF from Dihydro folate (DHF) must occur.

Many anti-cancer drugs act directly to inhibit thymidylate synthase, or indirectly, by inhibiting DHFR.

The enzyme Dihydro folate reductase (DHFR) is a target of the anticancer drugs aminopterin and Methotrexate.

These drugs are analogs of DHF and act as competitive inhibitors of DHFR. Inhibition of this enzyme prevents the regeneration of THF and blocks dTMP synthesis because of the lack of the methyl donor required for the reaction of thymidylate synthase.

The class of molecules used to inhibit thymidylate synthase are called the suicide substrates because they irreversibly inhibit the enzyme. Molecules of this class include 5-fluorouracil and 5-fluorodeoxyuridine. Both are converted within cells to 5-fluorodeoxyuridylate, FdUMP. It is this drug metabolite that inhibits thymidylate synthase.

 Thymidylate synthase

 

 Figure- 4-Thymidylate synthesized by the Methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase. Tetrahydrofolate (THF) is regenerated from the dihydrofolate (DHF) product of the thymidylate synthase reaction by the action of dihydrofolate reductase (DHFR), an enzyme that requires NADPH. Methotrexate by  inhibiting DHFR impairs the regeneration of THF which is the metabolically active form of folic acid. Folic acid deficiency and impaired synthesis of dTMP and thereby decreased DNA synthesis is the outcome of methotrexate therapy.

Cells that are unable to regenerate THF suffer defective DNA synthesis and eventual death. For this reason, as well as the fact that dTTP is utilized only in DNA, it is therapeutically possible to target rapidly proliferating cells over non-proliferating cells through the inhibition of thymidylate synthase.

Role of folic acid in purine synthesis

THF is also required as a donor of two carbon atoms in the synthesis of the purine ring structure required for adenine and guanine. The carbon atoms donated by THF are indicated in Figure -5.Therefore, a lack of THF blocks the synthesis of the purine ring structure because of the lack of the ability of the cell to synthesize N10-formyl-THF.

 Purine ring

Figure-5- showing origin of the atoms of the purine base.

In summary, DNA synthesis requires synthesis of dTMP and the purines adenine and guanine. THF, derived from the vitamin folic acid, is required for the biosynthesis of these nucleotides.

Adverse effects of methotrexate

Methotrexate blocks the cell’s ability to regenerate THF, leading to inhibition of these biosynthetic pathways. The lack of nucleotides prevents DNA synthesis, and these cancer cells cannot divide without DNA synthesis.

Unfortunately, the effects of Methotrexate are nonspecific and other rapidly dividing cells such as epithelial cells in the oral cavity, intestine, skin, and blood cells are also inhibited. This leads to the side effects associated with methotrexate (and other cancer chemotherapy drugs) such as mouth sores, low white blood cell counts, macrocytic anemia, gastrointestinal discomfort, hair loss, skin rashes, and itching. Less frequent adverse effects include reversible increases in transaminases and hypersensitivity-like pulmonary syndrome. Chronic low-dose methotrexate can cause hepatic fibrosis.

Hematologic side effects  include myelosuppression which is one of the primary toxic effects of methotrexate. Methotrexate suppressed hematopoiesis has been reported to have caused anemia, plastic anemia, pancytopenia, leukopenia, neutropenia, thrombocytopenia, lymphadenopathy, and lymphoproliferative disorders including reversible hypogammaglobulinemia (which has been reported rarely).

Antidote of Methotrexate

Leukemia patients are often given the compound Leucovorin (N5-formyl THF) following treatment with the drug methotrexate. Leucovorin (N5-formyl THF, folinic acid) is used as an antidote for cells that have decreased levels of folic acid. Treatment of leukemia patients with methotrexate kills the tumor cells but also other normal rapidly dividing cells. N5-formyl THF is normally administered 24 hours following treatment with methotrexate; it can be converted to THF by these normal cells by bypassing the block caused by methotrexate. Therefore, these normal cells can synthesize deoxy thymidine and carry out DNA synthesis.

Alternative Drug

Pemetrexed is a novel folate-directed antimetabolite. It is “multitargeted” in that it inhibits the activity of several enzymes, including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl iransferase, thereby affecting the synthesis of both purine and pyrimidine nucleic acid precursors. To avoid significant toxicity to the normal tissues, patients receiving pemetrexed should also receive low-dose folate and vitamin B12 supplementation. Pemetrexed has notable activity against certain lung cancers and, in combination with Cisplatin, also against mesotheliomas.

 

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Tumor markers

Tumor markers are hormones, enzymes, peptides or proteins abnormally synthesized and released by the cancer cells, or are produced by the host cells in response to cancerous growth.

Tumor markers may be present in the body fluids, blood, cell membranes or in the cytoplasm of the cell.

Clinical significance of tumor markers

Tumor markers are used as diagnostic and prognostic agents. They are used -

  • For screening of  cancer in asymptomatic individuals
  • As an adjunct in clinical staging of the cancerous condition
  • For monitoring during cancer treatment
  • For early detection of recurrence of the cancerous process.

Techniques of estimation

Tumor markers can be detected by -

  • Immunohistochemistry if they are present on cell membrane or in  cytoplasm of cell.
  • Radio immuno assay, Enzyme immuno assay or Immunochemical reactions if they are found in blood circulation.

Classification of tumor markers

The clinically important tumor markers are as follows-

 1) Tumor associated antigens- These are also called oncofetal proteins/antigens. The properties of cancer cells are changed and they start producing abnormal products from the mutated/ altered oncogenes.

The tumor associated antigens are produced in large amount in fetal life, but after birth these antigens disappear and are present in very minute amount in adults. In malignant cells, the synthesis of oncofetal antigens is reactivated and their concentration in blood and cancer cells in increased.

Examples of oncofetal antigens-

a) Carcinoembryonic antigen (CEA)- Gastrointestinal cancer, ovarian, breast, cervical and lung cancers , best marker of colorectal carcinoma.

b) Alpha feto protein (AFP)- Germ cell tumor and hepatocellular carcinoma

c) Tissue polypeptide antigen- Colonic cancer, breast and prostatic cancer.

 Other less important antigens are-

  • Pancreatic oncofetal antigen
  • Colon specific antigen
  • Beta oncofetal antigen

 2) Carbohydrate antigens

These antigens are more specific in determining the site of tumor. They are organ and tissue specific.

Examples of  important carbohydrate antigens are-

a) CA-125-Mainly ovarian cancer, but may also be elevated in endometrial cancer, fallopian tube cancer, lung cancer, breast cancer and gastrointestinal cancer.

b) CA-15-3- Breast cancer

c) CA-19-9-Mainly pancreatic cancer, but also colorectal cancer and other types of gastrointestinal cancers.

d) CA- 27-29- Breast cancer.

3) Pregnancy associated antigens

a) Human chorionic gonadotropin-β-subunits- (β-HCG)- It is a placental hormone, synthesized by the syncytiotrophoblastic cells of placental villi. In the non pregnant state it is present only in very minute concentration in the serum, but it is markedly elevated in pregnancy. The peak level is attained at 12 weeks of pregnancy, then it declines slowly to reach 1/5 th of peak at the end of 20 weeks and continues at a very low level for a few days even after parturition.

Measurement of HCG in serum and urine is undertaken to diagnose pregnancy.

Chemically it has two subunits-α and β-subunits.

The beta subunit is typically measured, because of its increased specificity and because some tumor cells secrete only Beta subunit.

Its high level is an ideal marker of gestational trophoblastic and germ cell tumors of testes and ovaries.

Its high level is also observed in Seminoma, embryonal carcinoma, teratocarcinoma and choriocarcinoma.

b) Placental like Alkaline phosphatase-(Regan isoenzyme- PLAP)- The Regan isoenzyme is a placental-type alkaline phosphatase that is expressed in a number of human tumors, particularly in gonadal and urologic cancers.

c) Other antigens

  • Human placental lactogen (HPL)
  • Sex hormone binding protein (SHBG)
  • Steroid binding β globulin (SBBG)
  • α2 Pregnancy associated globulin (PAG)

4) Mammary associated antigens

  • MCA (Mucin like carcinoma associated antigen)- Breast cancer
  • MAM- Breast cancer
  • MSA (Mammary serum antigen)- Breast cancer
  • MAP(Mitogen activated protein kinase)- Breast cancer

5) Hormones used as tumor markers

  • ACTH- Lung cancer, Medullary carcinoma of thyroid and pancreatic carcinoma
  • Calcitonin- Medullary carcinoma of thyroid
  • Catecholamines- Pheochromocytoma
  • Gastrin- Gastrinoma
  • Insulin- Insulinoma
  • Glucagon- Glucagonoma
  • Serotonin Carcinoid syndrome

6) Enzymes and Isoenzymes used as tumor markers

a)  Lactate dehydrogenase (LDH)

  • Total LDH- Lymphoma, leukemia, germ cell tumor, breast and lung cancer
  • LDH1- Germ cell tumors, ovarian carcinoma, Osteosarcoma
  • LDH 2,3,4- Leukemias
  • LDH-5- Hepatoma , breast cancer, colorectal cancer and other benign liver diseases

 b) Alkaline phosphatase (ALP)

  • Liver isoenzyme- Metastatic liver cancer
  • Bone isoenzyme- Metastatic bone disease, benign bone disease
  • Regan isoenzyme- Lung cancer, ovarian cancer, breast cancer, colonic cancer and uterine cancer
  • Nagao isoenzyme- Metastatic carcinoma of pleural surfaces, adenocarcinoma of pancreas and bile duct.

 c) Acid phosphatase

  • Prostatic acid phosphatase- Prostate cancer

 d) Creatine Kinase

  • CPK- BB- Adenocarcinoma, prostatic carcinoma

 e) α1- Antitrypsin- Germ cell tumor of testes and ovaries.

 f) Neuron specific Enolase- Neuroblastoma and Lung cancer

7) Miscellaneous tumor markers

  • Prostate specific antigen- Carcinoma prostate
  • Mono clonal immunoglobulins
  • Polyamines

Summary Table

S.no Tumor marker Significance
1. Carcinoembryonic antigen (CEA) Gastrointestinal cancer, ovarian, breast, cervical and lung cancers. Best marker of colorectal carcinoma. 
2. Alpha feto protein (AFP)  Germ cell tumor and hepatocellular carcinoma
3. Tissue polypeptide antigen.   Colonic cancer, breast and prostatic cancer
4. CA-125 Mainly ovarian cancer, but may also be elevated in endometrial cancer, fallopian tube cancer, lung cancer, breast cancer and gastrointestinal cancer
5.  CA-15-3  Breast cancer
6. CA-19-9 Mainly pancreatic cancer, but also colorectal cancer and other types of gastrointestinal cancers. 
7. β-HCG Gestational trophoblastic and germ cell tumors of testes and ovaries
8.  MAP(Mitogen activated protein kinase) Breast cancer 
9. ACTH Lung cancer, Medullary carcinoma of thyroid and pancreatic carcinoma 
10. Calcitonin  Medullary carcinoma of thyroid 
11. Catecholamines Pheochromocytoma 
12. Gastrin- Gastrinoma 
13. Insulin Insulinoma 
14. Glucagon- Glucagonoma 
15. Serotonin Carcinoid syndrome 
16. Total LDH Lymphoma, leukemia, germ cell tumor, breast and lung cancer 
17. Alkaline phosphatase (ALP)  Metastatic liver cancer, bone disease, lung and gonadal cancers 
18. Acid phosphatase Prostate cancer
19. Prostate specific antigen Prostate cancer

 

 

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Anticancer drugs

The goal of cancer treatment is first to eradicate the cancer. If this primary goal cannot be accomplished, the goal of cancer treatment shifts to palliation, the amelioration of symptoms, and preservation of quality of life while striving to extend life.

Cancer treatments are divided into four main types: surgery, radiation therapy (including photodynamic therapy), chemotherapy (including hormonal therapy and molecularly targeted therapy), and biologic therapy (including immunotherapy and gene therapy). The modalities are often used in combination, and agents in one category can act by several mechanisms. Surgery and radiation therapy are considered local treatments, though their effects can influence the behavior of tumor at remote sites. Chemotherapy and biologic therapy are usually systemic treatments.

Chemotherapeutic agents are used to treat various types of cancers. Although some are specific for cancer cells, most chemotherapeutic agents are toxic for both normal and cancer cells. When cure of cancer is possible, cancer treatments may be undertaken despite the certainty of severe and perhaps life-threatening toxicities.

Commonly Used Cancer Chemotherapy Agents

1) Direct DNA-Interacting Agents 

  • Cyclophosphamide
  • Mechlorethamine
  • Chlorambucil
  • Melphalan
  • Carmustine
  • Lomustine
  • Ifosfamide
  • Procarbazine
  • Cisplatin

2) Antitumor antibiotics 

  • Bleomycin
  • Actinomycin D
  • Mitomycin C
  • Etoposide
  • Doxorubicin and daunorubicin
  • Idarubicin
  • Epirubicin

3) Indirect DNA-Interacting Agents 

Antimetabolites 

  • 6-Mercaptopurine
  • 6-Thioguanine
  • Azathioprine
  • 2-Chlorodeoxyadenosine
  • Hydroxyurea
  • Methotrexate
  • 5-Fluorouracil
  • Cytosine arabinoside
  • Azacytidine
  • Asparaginase

4) Antimitotic agents 

  • Vincristine
  • Vinblastin

  5) Molecularly Targeted Agents 

  • Tretinoin
  • Bexarotene

 

Naturally occurring anticancer substances

1) Vitamin A and Beta Carotene

2) Vitamin E

3) Ascorbic acid

4) Selenium

5) Zinc

6) Quercetin- A flavonoids present in apple

7) Glucosinolates- present in bitter brussel sprouts

8) Epigallocatectin Gallate- present in green tea.

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Oncogenes in Human Cancer

  • Oncogenes are cancer susceptibility genes.
  • Proto-oncogenes are normal genes that are present in normal cells and are involved in normal growth and development.
  • Under certain circumstances due to the effect of certain processes proto oncogenes are converted to Oncogenes.

Significance of Proto oncogenes

In the normal cellular environment, proto-oncogene have crucial roles in cell proliferation and differentiation.(Figure 1) The normal growth and differentiation of cells is controlled by growth factors that bind to receptors on the surface of the cell. The signals generated by the membrane receptors are transmitted inside the cells through signaling cascades involving kinases, G proteins, and other regulatory proteins (See figure 2). Ultimately, these signals affect the activity of transcription factors in the nucleus, which regulate the expression of genes crucial in cell proliferation, cell differentiation, and cell death.  Proto-oncogene products have been found to function at critical steps in these pathways and inappropriate activation of these pathways can lead to tumorigenesis (Figure-1).

 Mechanism of oncogenesis

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure -1– Under the effect of Chemical carcinogens, radiations or viruses or in persons with genetic predisposition, the proto oncogenes are transformed to oncogenes. The oncogenes have either abnormal or more gene product resulting in altered cellular functions and malignant transformation of a normal cell to cancer cell.

Mechanisms of Oncogene Activation

Mechanisms that up regulate (or activate) cellular oncogenes (Proto-oncogenes) fall into four broad categories: point mutation, gene amplification, chromosomal rearrangement and insertional mutagenesis.

1) Point Mutation

Point mutation is a common mechanism of oncogene activation. For example, mutations in one of the RAS genes (HRAS, KRAS, or NRAS) are present in up to 85% of pancreatic cancers and 50% of colon cancers but are relatively uncommon in other cancer types. Most of the activated RAS genes contain point mutations in codons 12, 13, or 61.(figure 2)

Mechanism ofRasactivation- The gene product (p21) is related to G protein that modulates the activity of Adenylate cyclase and thus plays a key role in cellular responses to many hormones and drugs. p21 has GTPase activity also to terminate the hormonal action. The mutations in p21 appear to affect its conformation and to diminish its activity as a GTPase. The lowered activity of GTPase results in chronic stimulation of the activity of Adenylate cyclase, which normally is diminished when GDP is formed from GTP. The continuous  stimulation of  the activity of Adenylate cyclase can result in a number of effects on cellular metabolism exerted by the increased amount of c AMP affecting the activities of various cAMP dependent protein kinases. These events shift the balance of cellular metabolism towards a state favoring malignant transformation.

 Activation of RAS protein

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2- showing the effects of activates RAS protein. Growth factor- Receptor binding stimulates tyrosine kinase activity that stimulates, Ras protein , a G protein that is active when bound to GTP. Active Ras protein activates adenyate cyclase and a phosphorylation cascade is triggered. Mutated Ras protein remains active due to inability to replace GTP by GDP resulting in phosphorylation of cellular proteins and more synthesis of cell cycle regulatory proteins and thereby malignant transformation of the cell.

2) Gene Amplification

The second mechanism for activation of oncogenes is DNA sequence amplification (Figure-3), leading to over expression of the gene product.(Figure 3) Gene amplification is observed in tumors in patients on Methotrexate, an anticancer drug, an inhibitor of an enzyme dihydrofolate reductase. Tumor cells can become resistant to the action of this drug. The basis of this is that the gene for dihydrofolate reductase becomes amplified, resulting in an increase in activity of the enzyme up to 400 folds.

Certain cellular oncogenes can also be amplified in this manner and can get activated. Increased amount of the products of certain oncogenes, produced by gene amplification may play a role in the progression of tumor cells to a more malignant state.

Gene amplification 

 

 

 

 

 

 

 

 

 

 

Figure-3–showing gene amplification

3) Chromosomal Rearrangement

  • Chromosomal alterations provide important clues to the genetic changes in cancer.
  • The chromosomal alterations in human solid tumors such as carcinomas are heterogeneous and complex and likely reflect selection for the loss of tumor-suppressor genes on the involved chromosome.
  •  In contrast, the chromosome alterations in myeloid and lymphoid tumors are often simple translocations.
  • The basis of translocation is that a piece of one chromosome is split off and joined to another chromosome.
  • If the second chromosome donates material to the second, the translocation is said to be ‘Reciprocal’.
  • The breakpoints of recurring chromosome abnormalities usually occur at the site of cellular oncogenes.

Examples

a) Burkitt’s lymphoma, a B cell tumor characterized by a reciprocal translocation between chromosomes 8 and 14. The segment of chromosome 8 that breaks off and moves to chromosome 14 contains C-MYC.As shown in the figure-4, the transposition places the previously inactive C-MYC under the influence of the enhancer sequences the genes coding for the heavy chains of immunoglobulins. This juxta position results in activation of transcription of C-MYC.There is greatly increase synthesis of C-MYC coded DNA binding protein that acts to drive or force the cell towards becoming malignant, perhaps by an effect on the regulation of mitosis.

Enhancer activation by translocation, although not universal, appears to play an important role in malignant progression. In addition to transcription factors and signal transduction molecules, translocation may result in the over expression of cell cycle regulatory proteins such as cyclins and of proteins that regulate cell death such as bcl-2.

 Chromosomal translocation

 

 

 

 

 

 

 

 

 

Figure-4- showing chromosomal translocation in Burkitt’s lymphoma

b) Philadelphia chromosome

The first reproducible chromosome abnormality detected in human malignancy was the Philadelphia chromosome detected in CML (Chronic myelogenous leukemia). This cytogenetic abnormality is generated by reciprocal translocation involving the ABL oncogene, a tyrosine kinase on chromosome 9, being placed in proximity to the BCR (breakpoint cluster region) on chromosome 22. (Figure -5). The consequence of expression of the fused BCR-ABL gene product is the activation of signal transduction pathways leading to cell growth independent of normal external signals.

Normally C-ABL encodes a protein kinase. The juxta position results in chimeric BCL-ABR m RNA, which encodes a fusion protein displaying the increases tyrosine kinase activity. The increased activity transforms the normal cell to leukemic cell.

Imatinib, a drug that specifically blocks the activity of BCR-ABL has shown remarkable efficacy with little toxicity in patients with CML.

 Philadelphia chromosome

 

 

 

 

 

 

 

 

 

Figure-5- showing Philadelphia chromosome in Chronic Myelogenous Leukemia, Chromosome 9 and 22 are involved in this translocation forming BCR-ABL fusion gene.

4) Insertional mutagenesis- This process occurs in viral Oncogenesis. Certain viruses lack oncogenes, but may cause cancer over a longer period of time. When these viruses infect cells, a DNA copy (c DNA ) of their genome is synthesized by the activity of reverse transcriptase enzyme and the cDNA is integrated in to the host genome. The integrated double stranded c DNA is called “Provirus”. Based on the site of their insertion, two mechanism are involved –

a) Promoter Insertion– The cDNA copies of retroviruses are flanked at both ends by sequences named as long terminal repeats. These sequences are important in proviral integration and they can act as promoters of transcription (Figure-6).

For example-following infection of chicken B lymphocytes by certain avian leukemia viruses, the provirus becomes integrated near the myc gene. The myc gene is activated by an upstream, adjacent long terminal repeat acting as a promoter, resulting in transcription of its product in such cells. A B cell tumor is formed. By a similar mechanism human C-MYC gene is activated causing colorectal carcinoma.

 insertional mutagenesis

 

 

 

 

 

 

 

 

 

 

Figure-6- Proto-oncogene activation by insertional mutagenesis of MMTV-(a) Insertion of viral genomic DNA into somatic cellular DNA in close proximity of a silent oncogene. (b) Inserted proviral DNA induces the transcription of the oncogene.

b) Enhancer Insertion- In some cases the provirus is inserted downstream from the myc gene, or upstream from it but oriented in the reverse direction, nevertheless the myc gene becomes activated. Such activation can not be due to promoter insertion, since a promoter sequence must be upstream of the gene whose transcription it increases and the and the sequence must be in the correct 5’-3’ direction. Instead the enhancer sequences present in the long terminal repeat sequences of the retroviruses are involved.

Of the mechanisms described above, the mechanisms like-promoter insertion, enhancer insertion, chromosome translocation and gene amplification, cause an increase in the amount of the gene product due to increased transcription of the oncogene. Thus, an increase amount of the product of an oncogene may be sufficient enough to push a cell towards becoming malignant.

conversion of proto oncogenes to oncogenes

 

 

Figure-7- Summary of the mechanisms involved in the conversion of Proto oncogenes to Oncogenes. If the point mutation takes place in the regulatory genes, excess gene product is formed but if the point mutation takes place in the structural genes, abnormal gene product is formed.

Point mutation, on the other hand involves a change in the structure of the gene product, but the amount mostly remains the same unless it is in the regulatory genes (Figure-7).Thus it implies that the presence of a structurally abnormal key regulatory protein in a cell may also shift the equilibrium of a cell towards malignant transformation.

Activation of oncogenes alone is not the sole pathway of malignancy. A combination of activation of oncogenes and inactivation of tumor suppressor genes is involved in the malignant transformation of cells  in certain types of cancers(Figure-8).

Oncogenes versus Tumor suppressor genes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-8- Highlighting the role of oncogenes and tumor suppressor genes in carcinogenesis

Product of oncogenes

1) They act on key intracellular pathway involved in growth control.

2) They act as DNA binding proteins to affect the control of the cell cycle

3) The product of certain oncogenes act as growth factors or imitate the action of an occupied growth factor receptors.

 

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Cancer susceptibility genes

There are two major classes of cancer susceptibility genes.

A) Genes affecting cell growth- These genes exert their effects on tumor growth through their ability to control cell division (cell birth) or cell death (apoptosis).There are two types of genes affecting cell growth.

  • Tumor suppressor genes- negatively affect cell growth. The normal function of tumor-suppressor genes is to restrain cell growth, and this function is lost in cancer. Because of the diploid nature of mammalian cells, both alleles must be inactivated to completely lose the function of a tumor-suppressor gene, leading to a recessive mechanism at the cellular level.
  •  Oncogenes- directly affect positive cell growth. Oncogenes are tightly regulated in normal cells, they are called proto oncogenes. In cancer cells, proto oncogenes acquire mutations that relieve this control and lead to increased activity of the gene product.

B) Care taker genes-The second class of cancer genes, the caretakers, do not directly affect cell growth but rather affects the ability of the cell to maintain the integrity of its genome. Cells with deficiency in these genes have an increased rate of mutations in all the genes, including oncogenes and tumor-suppressor genes.

Tumor suppressor genes

Tumor suppressor genes are also called “Anti oncogenes or recessive oncogenes”. They act differently from oncogenes in that their inactivation (as opposed to activation of oncogenes) removes constraints on control of growth.

Differences between oncogenes and tumor suppressor genes

Characteristics Oncogenes Tumor suppressor genes
Mutations Mutations in one of the two alleles is sufficient for activity Mutations in both of the alleles has to be there
Functions of a protein There is “gain of function” of a protein that signals cell division Loss of function of a protein. There is loss of regulation.
Inheritance Mutations arise in somatic tissues thus they are not inherited Mutations present in germ cells can be inherited.
Tissue preference Some tissue preference is observed Strong tissue preference.

Examples of tumor suppressor genes

1) RB1 gene- This gene is involved in the formation of Retinoblastoma, a malignant tumor of retinal neuroblasts, which are precursor cells of photoreceptor cells in the retina. In some cases, the tumor is inherited, while in others it does not appear to be hereditary in nature.

Function of RB1 gene product

The protein product of RB gene (pRB) is expressed in many cells.

  • It is a nuclear protein to regulate the cell cycle.
  • It binds to certain viral proteins to inactivate them.
  • It also binds to certain transcription factors that are active in the S phase of the cell cycle and thus slows cell cycling.
  • Mutations in the genes are responsible for Retinoblastoma, Osteosarcoma and certain other human tumors.
  • In hereditary case of retinoblastoma, the first mutation exists in the germ cell lines and the second is acquired during the life time in retinoblasts. This phenomenon is called “Loss of heterozygosity”. Since initially before mutations the individuals were heterozygous in the region of RB gene- one normal and other mutated allele.
  • Knudson hypothesis postulates that the development of retinoblastoma depends upon two mutations
  • In sporadic (Non hereditary cases) both mutations occur in the retinoblasts, both are acquired during the life time of an individual.

2) BRCA-1

Tumor-suppressor gene, BRCA-1, has been identified at the chromosomal locus 17q21.

Functions of BRCA-1 gene product

  • This gene encodes a zinc finger protein (DNA binding protein with a special motif to exclusively bind DNA) and the product therefore may function as a transcription factor.
  • The gene product also appears to be involved in gene repair.
  • Cells defective in BRCA1 possesses numerous cytological and biological features that have been correlated with perturbation in the maintenance of chromosome stability.
  • BRCA1 has been shown to function in various transcriptional mechanisms, suggesting that the function of this important protein may go well beyond its well-documented role in Double Strand Break repair.
  • BRCA1 has been reported to interact with as many as 50 proteins.
  • Women who inherit a mutated allele of this gene from either parent have at least a 60–80% lifetime chance of developing breast cancer and about a 33% chance of developing ovarian cancer.
  • Men who carry a mutant allele of the gene have an increased incidence of prostate cancer and breast cancer.

3) BRCA-2,

 This gene has been localized to chromosome 13q12; it is also associated with an increased incidence of breast cancer in men and women.

4) P53 gene

The p53 mutation is present in nearly 40% of human breast cancers as an acquired defect. The p53 tumor suppressor gene acts as the ‘Guardian of the Genome’. It encodes a protein of molecular weight 53 kDa, This protein is nuclear in location and is subjected to phosphorylation and dephosphorylation. The p53 has three major effects-

  • It acts as a transcriptional activator regulating certain genes involved in the cell cycle
  • It acts as G1 check point control for the DNA damage. If excess damage to the DNA has occurred, it causes inhibition of the cell cycle, allowing time for repair. If the cell cycle proceeds without repair DNA damage would be replicated, introducing permanent mutations in to the genome. If the p53 is inactivated or mutated as happens in certain tumors, DNA damage accumulates and the DNA becomes unstable.
  •  p53 also participates in apoptosis. The purpose of this function is that it hastens the death of the potentially dangerous cells e.g. those damaged by UV light which have the potential to become cancer cells.
  •  Besides all this p53 also combines with certain viral proteins.
  • Mutations in the p53 gene are the most common genetic alterations in human cancer and are frequent in Breast, colon and lung cancers.

5) PTEN

  • Acquired mutations in PTEN occur in about 10% of the cases.
  • The gene appears to be located on chromosome 10 and appears to be an important ‘tumor suppressor’ and when mutated it allow the cells to grow out of the control and become malignant.
  • This gene is also called “MMAC1”, Mutations in this gene also play an important role in the aggressiveness of the tumor.

 

 

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“International Union Against cancer” has defined Cancer as a disturbance of growth, characterized by excessive proliferation of cells without apparent relation to physiological demands of the organ involved.

Properties of cancer cells

Three important properties-

1) Diminished or unrestricted control of growth

2) Capability of invasion of local tissues

3) Capable of spreading to distant parts of body by metastasis.

Characteristics of cancer cells (Deviations from normal characteristics)

1) Morphological changes

  • Round shape, larger than normal
  • Altered nuclear : cytoplasmic ratio
  • Transformed cells grow over another and form multilayers.
  • Can grow without attaching to the surface (in vitro), diminished adhesion

2) Biochemical Changes

  • Increased synthesis of DNA and RNA
  • Increased rate of glycolysis- both aerobic and anaerobic
  • Show alteration of permeability and surface charges
  • Alteration of oligosaccharide chains
  • Increased activity of Ribonucleotide reductase and decreased catabolism of pyrimidines
  • Alteration of isoenzyme pattern to fetal proteins
  • Appearance of new antigens and loss of certain antigens
  • Changes of glycolipid  and glycoprotein constituents on cell surface
  • Alteration of the activities of certain enzymes such as proteases
  • Alteration in transport properties
  • Inappropriate synthesis of certain hormones and growth factors.

 

Etiology of cancer

Agents causing cancer fall in to three broad groups-

a) Radiant energy

b) Chemical compounds

c) Viruses

There may also be familial causes due to mutation in specific genes (e.g. tumor suppressor genes)

a) Radiant energy

  • UV Rays
  • X-Rays
  • Y-Rays

 Mechanism of carcinogenesis by radiations- Damage to DNA is the basic mechanism

  • UV Rays cause the formation of pyrimidine dimers, apurinic or apyrimidinic sites, single or double strand breaks or by causing cross linking of strands.
  • X-rays and Y-Rays, apart from causing direct damage to DNA, cause generation of free radicals also.The resultant free radicals interact with DNA and other macromolecules, leading to molecular damage contributing to carcinogenic effect of radiant energy.

b) Chemical Compounds

A wide variety of chemicals are carcinogenic. Some of these are direct reacting and majority occurs as procarcinogens which are converted in the body to ultimate carcinogenic chemicals. These chemicals gain entry in to the body through diet, environment (occupation, life style) or through drugs used for a therapeutic cause.

 Examples of chemical carcinogens

S.No. Class Compound
1. Polycyclic aromatic hydrocarbons Benzo(α) pyrene, Dimethyl benzanthracene
2. Aromatic amines 2-Acetyl amino fluorine,N-Methyl-4 amino azo benzene
3. Nitrosamines Dimethylnitrosamine, Diethyl nitrosamine
4. Drugs Alkylating agents, diethyl stibestrol
5. Naturally occurring compounds Dactinomycin, Aflatoxin B
6. Inorganic compounds Arsenic, asbestos, beryllium, cadmium, chromium, nickel, vinyl chloride, β- propiolactone etc.

Mechanism of action of chemical carcinogens

  • Some may be direct acting- β- propiolactone, methyl cholanthrene; these agents interact directly with the target molecule.
  • Some require metabolic activation- Aromatic hydrocarbons, aromatic amines etc.

Metabolic activation- The process whereby one or more enzyme catalyzed reactions convert procarcinogens to active carcinogens is called metabolic activation. Any intermediate compounds formed are called proximate carcinogens. The sequence can be displayed as follows-

 Procarcinogen—–> Proximate Carcinogen—–>Ultimate carcinogen

The Procarcinogen in itself is not a chemically reactive species, whereas the ultimate carcinogen is highly reactive. The procarcinogens are electrophiles (molecules deficient in electrons), which readily attack nucleophilic (electron rich) groups in DNA, RNA and proteins.

The metabolism of procarcinogens involves action by mono-oxygenases (cytochrome P450) and transferases. The activities of these enzymes are affected by a number of factors- such as species, age, gender and genetic variations. The variations in activities of these enzymes help explain the often appreciable differences in carcinogenicity of chemicals among different species and different individuals of the same species.

Damage to DNA- can be by

1) Covalent binding- The carcinogens and their derivatives bind covalently to cellular molecules such as DNA, RNA and proteins.

2) These agents interact with the purines, pyrimidines or Phospho diester groups of DNA.  Most common site of attack is guanine.

The covalent interactions of direct or ultimate carcinogens with DNA can result in several types of DNA damage. This damage can be repaired by the repair system. The unrepaired damage leads to mutations.

c) Viral Oncogenesis

The Oncogenic viruses contain DNA or RNA as a genome.

i) DNA Viruses

Many DNA viruses cause tumor in animals. The DNA viruses causing cancers in human are-

  • Epstein- Barr virus-causes Burkitt’s lymphoma and nasopharyngeal carcinoma.
  • Hepatitis B Virus- is probably the major etiological agent for many primary liver cancers.
  •  Human Papilloma virus (HPV)- Multiple warts and cervical cancer.

Mechanism of action of DNA viruses

  •  DNA viruses bind tightly to the DNA and alter the gene expression.
  • The viral proteins show co-operative effect suggesting that alteration of more than one reaction takes place for malignant transformation.

 ii) RNA Viruses (Retro viruses)

  • These viruses convert their RNA genome in to DNA with the help of reverse transcriptase enzyme,
  • The resultant DNA is integrated in to the DNA of normal cells causing malignant transformation by various mechanisms.
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