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Introduction to Apoptosis
Apoptosis is a co-ordinated and apparently internally programmed process that mediates the death of cells in a variety of biologically significant situations viz. cytotoxic T-cell killing, atrophy in response to withdrawal of endocrine and other stimuli, etc. Identical changes are observed in normal and tumour tissues exposed to low or moderate doses of chemotherapeutic agents or to ionising radiations. Biochemical events lead to characteristic cell changes (morphology) and death: These changes include Blebbing, Cell shrinkage, Nuclear fragmentation, Chromatin condensation, Chromosomal DNA fragmentation and Global mRNA decay.
Structural and Morphological Changes includes: Dramatic shrinkage of cell volume, accompanied by dilatation of ER and convolution of the plasma membrane. The cells break up into membrane bound spherical bodies containing structurally normal but compacted organelles. Nucleus undergoes profound chromatin condensation around the nuclear periphery. Nucleolus falls apart, nuclear pores appear to aggregate
Biochemical and Molecular Changes in Apoptosis: In Biochemical Changes several cytoskeletal proteins and the nuclear lamins undergo site specific proteolysis. Activation of transglutaminase results in protein-protein cross-linking. Nuclease activation cleaves first large and then small oligonucleosomal fragments of chromatin.
The Effector Pathway of Apoptosis:
1. Role of ICE Family Proteases: A family of cysteine proteases exhibit preference for cleavage adjacent to aspartate residue, mammalian cells contain about 10 distinct members one of which is Interleukin-1β - converting enzyme (ICE). The nuclear protein Poly ADP-ribose Polymerase is consistently cleaved and inactivated early in apoptosis by the ICE family protease CPP 32.
2. Role of Cytokines: Cytokines play an important role in the signalling of cell death. “CD-95/apo-1/fas” is a transmembrane receptor belonging to the receptor family “CD-40/TNF” receptor. “Fas” signalling is now considered to be one of the key events in CTL killing.
3. Role of Ceramide: Ceramide has been incriminated as signal for activation of apoptosis that is released from membrane lipids on digestion by “sphingomyelinases”.
4. Role of Cytotoxic T-cells: Cytotoxic T-cells also possess granules that release a variety of effector proteins, e.g. Perforin & Granzyme B.
5. Role of P53 and interferon-response factor-1 (IRF-1): P53 and IRF-1 are both onco-suppressor genes that signal DNA injury. Both oncosuppressors can induce apoptosis in the appropriate circumstances.
Role of Apoptosis in Carcinogenesis
Abrogation of apoptosis is a general mechanism in carcinogenesis. Many non-viral carcinogenic agents also work through survival and replication of cells bearing mutations. Mutations arise through double-strand breaks, e.g. after ionizing radiation or single-strand break (after UV rays) or bulky adducts after exposure to many chemical carcinogens. Failure in apoptosis may permit clonal outgrowth of cells with inappropriate recombination events resulting to tumor
Relationship of Apoptosis and Tumor Suppressor Genes: DNA damage, if not repaired, may stimulate and initiate apoptosis, the degree of which vary from tissue to tissue. If oncogenic suppressor gene P53 is simultaneously absent the mutagenic effect progresses to cancer.
Metastasis is the spread of cancer cells from the primary site of origin to other tissues, both neighbouring and distant, where they grow as the secondary tumours. Benign tumours can grow very rapidly and attain big sizes and may be sometimes life-threatening, but they do not metastasize. It is the malignant tumours, cancerous ones, invade surrounding tissues and send out cells to begin new tumours at distant sites. The spread may be bloodborne/or through lymphatics. This colonization at distant sites is metastasis and is the major cause of death from human malignancies. Tumour cells must attach to degrade and penetrate the “extracellular matrix” (ECM) at several steps of metastasis. Approximately 50 per cent of patients who develop malignant tumors can be cured with various therapies, viz. surgical removal, radiation therapy and chemotherapy. Of the remaining 50 per cent , majority die because of metastasis.
Biochemistry of Metastasis
In the third section of lecture educator explains the Biochemistry of Metastasis starting with the explanation of Interaction of Invading Cancer Cells with ECM. Extracellular matrix (ECM) can be divided into two major categories: Basement membrane (BM) and Interstitial connective tissue (ICT).
Important Constituents of ECM are Collagen (Basement membrane contains type IV collagen and interstitial connective tissues type I and type III collagen) and Adhesion-promoting proteins (Basement membrane contains laminin and interstitial connective tissue) fibronectin ( Both laminin and fibronectin are large multifunctional molecules that can bind to other ECM components such as collagen, proteoglycans and to cells, Attachment of cells to laminin and fibronectin is brought about by distinct and specific cell surface receptors).
Basement membrane (BM) is the first tough elastic barrier that surrounds both tissues and blood vessels. Interaction of Invading Cancer Cells with ECM has three steps. In step 1- The tumour cell binds to one of the membrane’s glycoproteins, a cross-shaped molecule called as laminin and Laminin appears to serve as a bridge between receptors on the surface of the invading cancer cell and the BM itself. In step 2 Once the tumour cell is attached to laminin, the invasive tumour cell secretes certain proteolytic enzymes that degrade the BM. In step 3 Factors that favour migration of tumour cells in the passage created by the degradation of EC matrix including BM are not well understood.
In this section educator explains the Oncogenic Markers or Tumour Markers. Tumour “markers” are defined as a biochemical substance (e.g. hormone, enzymes, or proteins) synthesized and released by cancer cells or produced by the host in response to cancerous substance and are used to monitor or identify the presence of a cancerous growth. Tumour markers may be present in Blood circulation, Body Cavity fluids, Cell membranes and Cell cytoplasm. Tumour markers are different from substances produced by normal cells, in quantity and quality.
Immunohistological and immunocytological tests are used to detect those tumour markers which are present only on cell-membranes and cytoplasm of cells and not in blood circulation, also Biochemical methods are used for measuring tumour markers found in the blood circulation.
There are two Types of Tumour Markers 1. Tumor-Specific Antigens: These are a direct product of oncogenesis induced by an oncogene (viral), radiation, chemical carcinogen or an unknown risk factor. 2. Tumor-Associated Antigens: Also called as oncofoetal proteins/antigens, shown to exist in both in embryo-foetal tissues and cancer cells, these are produced in large quantities in foetal life and released in foetal circulation. With the onset of malignancy in adult life, the synthesis of oncofoetal antigens in foetal life, is again reactivated with malignant transformation of cells and reappears in cancer cells and in blood circulation (Retrogenetic expression theory).
Tumor Marker: Characteristics & Clinical Significance
An ideal tumour marker is yet to be found out. Ideal tumour marker must satisfy the following criterias:
(a) Analytical Criteria: they should have high sensitivity; the test method should be sensitive to measure very low concentrations. They should have high specificity, it should measure a particular tumour marker only and no other substances should interfere. They should have high accuracy. They should have high precision and method should be simple and easy to measure.
(b) Clinical Criteria: they should be disease-sensitive, should have high disease-specificity, should be stable and should not show wide fluctuations, should correlate well with the cancerous process, i.e. Its extent and the volume of the tumour, should correlate well with cure rate, should prognosticate the ‘high risk’ cancer patients from “lower risk”, should be able to detect relapse/recurrence of the cancer.
Commonly Used Tumor Markers are 1. Carcinoembryonic Antigen (CEA), CEA is one of the oncofoetal antigens, Gold and Freedman as a tumour specific antigen present only in cancer cells, it was discovered in 1965 by raising antiserum against a colon cancer. 2. Human Chorionic Gonadotropin (β-HCG), HCG is a placental hormone. It is synthesised by the syncytiotrophoblastic cells of placental villi, present in the serum of nonpregnant women in trace amounts or not at all, markedly elevated in pregnancy and Measurement of elevated HCG in serum and urine has been used to diagnose pregnancy. 3. Alpha-Fetoprotein (AFP), AFP is synthesised in the liver, yolk sac and GI tract in foetal life and is released into the serum of foetus, it is a normal component of serum protein in human foetus. The concentration is highest during embryonic and foetal life. At birth, the serum AFP declines to 1/ 100th of AFP value, at one year of life, the value decreases further and in normal adults it is less than 20 ng/ml
Tumour Markers not Commonly Used are Tissue polypeptide antigen (TPA), Tennesee Antigen (Tenagen), CA 125, CA 15-3 and CA 19-9.