18.

Cell Ageing and Cell Death

In the cells life there are three phases: growing with normal metabolic activity, ageing and death. During ageing, cells volume and mitotic rhythm decreases, ratio of death cells in tissues increases, nucleolo-nuclear and nucleo-cytoplasmic ratios diminish, and nuclear changes appear: shrinkage and condensation of nuclei (pycnosis, and hyperchromia) nuclear fragmentation (karyorrhexis) and dissolution of nucleus (karyolysis). In cytoplasm, basophylia decreases, pigments and lipids accumulate, and the number of vacuoles increases. The death is instantaneous. Post-mortem the cell is round, pseudopodia are retracted, mitochondria swell and disappear. There are two types of cell death: necrosis and apoptosis. Necrosis is an accidental death occurring in anoxia (in cerebral, myocardial, or renal stroke), in thrombosis, after burns or freezing, etc. In necrosis, lysosomal enzymes activate inside the cytoplasm and begin to digest the cellular structures. The cell swells and eventually plasma membrane breaks. Consequently, the lysosomal enzymes can be released outside the cell, together with other cellular molecules resulting in local inflammation that can also affect the surrounding cells. Apoptosis was described as a physiological death after the cell lived an appropriate life time. Apoptosis is another type of cell death occurring according to a totally different mechanism. In this process lysosomes are involved only in the last stages of the cell death. Unlike the necrosis, when the protein synthesis is arrested, in apoptosis specific proteins are produced that are important in the organized breaking apart of the cellular structures. In apoptosis specific proteases are involved, that are stepwise activated in cascade. The most important enzymes for apoptosis are known as caspases (cystein- asparagil proteases). Some of them are activated by extracellular signals such as Fas polypeptide released by killer lymphocytes that attaches to specific receptors on the cells surface (death receptors) that trigger intracellular changes specific for apoptosis. Other caspases are activated by intracellular signals such as cytochrome c released from swollen mitochondria, after the outer mitochondrial membrane permeabilisation. The activated caspases by either of the above mentioned mechanisms attack the cytoskeleton the cells becomes round, and the nuclear lamina nuclear changes. One of the earliest phenomena is also DNA degradation to fragments of 200 base pairs or multiples of 200 bp. In apoptosis cell does not swell, by the contrary it shrinks and there is no inflammation on site, since the apoptotic cell is phagocytosed by macrophages or by the neighbouring cells.

Apoptosis is important during embryogenesis, metamorphosis, differentiation, and in cellular turn-over in the different tissues. Cytostatic drug cisplatin triggers the malignant cells apoptosis, and the AIDS virus HIV produces the apoptosis of the immune T lymphocytes. There are different theories of cell ageing. Theory of errors explains the cellular ageing by accumulation of errors in DNA during replication, until the cell can not function. Another one is theory of integrated virus that is activated at a certain moment. These theories can not explain the different life time for different cells types. Theory of programmed cell death postulates that each cell has its own genetic program controlling a certain duration of life, after which the apoptosis mechanisms are activated. Hayflick demonstrated on cultured skin fibroblasts that these cells maintain their specific number of cycles, regardless the renewal of culture medium. Embryo fibroblasts have 50 cell cycles, while fibroblasts from 10 years old child have 48 cycles. He concluded that the number of cell cycles performed by fibroblasts decreases with 0.2/year of age. If the cell culture is frozen in liquid nitrogen, after unfreezing, fibroblasts resume exactly the remaining number of cycles. This way, the effects of radiation or of toxic compounds accumulation are excluded. The conclusion was that the fibroblasts are programmed for 50 cell cycles. These findings have important medical application such as in cancer therapy. It is known that malignant cells are immortal, and the elucidation of apoptosis mechanisms can allow the triggering of apoptosis in malignant cells. The theory of programmed cell death is a materialistic one, since the duration of human life is not programmed, only the duration of the cells life is programmed. The cell ageing cannot be extrapolated to human ageing. The cell ageing is the decrease in capability of cells to divide in culture; the human or animal organism ageing is related with an increased probability of cell death in a certain interval of time. Therefore it remains possible, at least in theory, to extend the duration of human life.

19. Central Dogma of Molecular Biology and the Medical Applications

19.1 Central dogma of molecular biology

Central dogma of molecular biology is a representation for the flux of genetic information in the cell:

DNA
auto-replication

transcription

RNA

translation

Proteins

The central dogma is valid in all prokaryotic and eukaryotic cells. In eukaryotic cells genetic material located inside nucleus. DNA replication and mRNA synthesis occur in nucleus, after that RNA passes through the nuclear pores in cytoplasm, where proteins are synthesized by ribosomes. Prokaryotic cells dont have nucleus and all these processes occur in cytoplasm.

19.2 Genetic material

Genetic material is the material support of heredity and it consists of DNA in both eukaryotic and prokaryotic cells. In eukaryotes there are linear DNA molecules in chromosomes and circular DNA molecules in mitochondria; chloroplasts in plants also contain circular DNA molecules. DNA contains genes in which the genetic information is encoded in the nucleotide sequence. The total number of chromosomal genes represents the genome. The human nuclear genome is estimated to contain 20,000-25,000 genes, comprising only about 2% of the entire DNA. The remaining chromosomal DNA consists of non-coding regions, with other functions including in gene regulation. The human mitochondrial genome contains 37 genes. DNA in prokaryotes has circular shape and is included in two genetic elements: chromosome (a big DNA ring), and plasmids (smaller DNA rings). In the prokaryotic chromosome there are several thousands of essential genes for cell growth and multiplication. The plasmids contain 3-200 optional genes that increase the possibilities of bacteria to survive in difficult conditions. An exchange of genes between chromosome and plasmids is possible, even the entire plasmid may be integrated in the chromosome and in this case is named episome.
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The plasmids have a high medical importance. On one hand, they can be used as vehicles in recombinant DNA technology. The plasmids are extracted from bacteria through cell lysis followed by ultra-centrifugation. New genes are inserted inside them; the modified plasmids are next introduced in other cultured bacteria which will synthesize the proteins encoded by the foreign genes. Finally, the protein of interest is purified from the culture medium. Many such proteins of great medical interest are produced through this technology, and used in practice: interferons (proteins synthesized by cells infected with viruses and released in the environment to protect other cells against the viral infection), human insulin, human growth hormones, anti-haemophilic protein and erythropoietin. A second reason of medical interest for plasmids is given by their genes that provide resistance to antibiotics.

19.3 Essentials of DNA replication

In order to understand the replication is important to know the DNA structure. Genes consist of several hundreds or even thousands of nucleotides as structural units of DNA. A nucleotide is made of a nitrogenous base that can be a purine (adenine and guanine) or pyrimidine (tymine and cytosine), a pentose (deoxyribose) and a phosphoric acid. Thus the nucleotides differ to one another by the nitrogenous base. In nucleic acids, the nucleotides are linked through phospho-di-estheric bonds between the pentoses of neighbouring nucleotides. A DNA molecule is formed by two chains of polynucleotides linked by hydrogen bonds between the purines and pirymidines from the two chains. The nucleotide sequence on the second strand (chain) is determined by the sequence of nucleotides from the first strand, because they are complementary and base pairs are formed: A=T, G C. The DNA molecule is a double helix (duplex), which was compared with spiral stairs in which nitrogenous bases are the stairs and deoxyriboses linked through phospho-di-estheric bonds are the banisters. The chains are anti-parallel, because the direction of the phospho-di-estheric bonds on one chain is opposite as compared to that of the paired chain. One end of chain has the OH group from the 3 position free (named the 3 end) and at the same end of molecule, the opposite chain has the OH group from position 5 free (the 5 end). When the DNA replicates and the quantity of the genetic material is doubled, the bases sequence is conserved. The DNA duplex splits into the two strands and on each strand a new complementary strand is synthesized. By the separation of the old strands replication fork is formed, and the synthesis of the DNA is performed by enzymes named DNA polymerases. These enzymes work in 5 3 direction only. For this reason, the synthesis on the old strand with the 3 end free is
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continuous and more rapid than on the opposite strand and this first strand is called leading strand. On the opposite strand, with the 5 end free, the synthesis is not continuous, and is performed in the opposite direction of the replication fork, in fragments named Okazaki fragments. Finally, the Okazaki fragments are linked by DNA ligase and will form the second new strand, but because the synthesis is slower than on the leading strand, the second old strand, is named lagging strand. After the DNA synthesis is completed, the duplexes formed from one old and one new strand separate. For this reason, the replication is semi-conservative (aspect demonstrated by Meselson & Stahl using N15). In the chromosome of Escherichia coli, a single replication origin is seen with the electron microscope, from which two replication forks develop in the opposite directions. When the forks meet, the replication finishes and two chromosomes will separate. During the cell division the DNA is equally divided to the daughter cells, and each cell receives the same genetic information like the mother cell, the transmitted genetic material being the support of heredity. There are some peculiarities of replication in eukaryotes. 1. Speed of replication is 10 times smaller in eukaryotes than in prokaryotes (50 nucleotides/second versus 500 nucleotides/second in prokaryotes); since a human chromosome has around 150 millions nucleotides, with a single origin of replication around 800 hours would be necessary for the entire replication. But the replication needs to be finished during the S phase of the cell cycle, in 7-8 hours and this is realized using more replication origins on each chromosome (around 100 on each human chromosome). In each origin two replication forks appear and go in the opposite directions. 2. Okazaki fragments in eukaryotes are shorter (100-200 nucleotides) than in prokaryotes (1,000 to 2,000 nucleotides) because of the organisation of chromatin in nucleosomes. 3. Origins of replications appear in groups of 20-80, situated in a certain region of chromosome; each group is named replication unit or replicon. The replicons are gradually activated during the S phase, and the heterochromatin regions are the last activated. 4. Synthesis of histones is performed in parallel with the DNA replication only in the S phase. During the replication, the protein core of old nucleosomes remain attached to one DNA duplex, and on the second duplex only newly synthesized cores are linked. 5. DNA polymerases in eukaryotes are different as compare to those of prokaryotes.

19.4 Medical implication and applications from the study of DNA replication

The main medical implications and applications are mutations and inhibitors of DNA synthesis. Preservation and correct transmission of the genetic information involve a precise and correct replication of DNA that is the accurate reproduction of the base sequence from the parental molecule in the daughter molecules. Errors occurred in replication drive to appearance of mutations. A certain level of mutations (generally low) was necessary for the evolution of species. Over a certain limit, the mutations are dangerous and incompatible with the cells life or drive to uncontrolled proliferation of cells (cancer). For this reason the cells have mechanisms for reparation of errors that can appear spontaneously in DNA or that are induced by chemical or physical agents. The mutations can involve one nucleotide and are named point mutations or more complex, like the absence of some DNA sequences, named deletions, or presence of extra sequences named insertions. The point mutations can appear spontaneously in DNA. For example, deamination through hydrolysis transforms the cytosine in uracyl, and during the replication, the G C pair will be replaced with A=T, because the uracyl is complementary only with adenine. Depurination can also take place through spontaneous hydrolysis, and means that an adenine or a guanine is removed from DNA. This is the most frequent lesion of DNA; around 5,000 purines are lost every day in each human cell, comparative with 100 deaminations. Reparation of lesions produced through deamination consists in removal the defective base through hydrolysis. This step is performed by enzymes named DNA glycosylases; there are around 20 enzymes in this group, each one specific for a certain base change. After the action of glycosylases, in DNA a lesion similar with the depurination remains: a base is absent. The region with the missing base is excised by another enzyme (an endonuclease), a new correct fragment is synthesized by the reparatory DNA polymerase, and finally the DNA ligase links the new fragment with the old one on the strand. The mutations induced by chemical or physical agents may appear with a higher frequency in DNA. These mutagenic agents are in the same time and in almost all case carcinogenic. Chemical mutagens. Some chemicals produce direct changes of DNA bases. Alkylating agents insert alkyl radicals (methyl, ethyl) in DNA. They are carcinogenic, but in the same time the malignant cells are more sensitive to these agents. The normal cells can repair the lesions produced by alkylating agents, but the malignant cells have deficient reparatory mechanisms. Some alkylating agents like the compounds derived from nitroso-urea are used in the chemical therapy of cancer.
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Other mutagenic agents intercalate between the DNA base pairs and produce distortions of duplex, which drive to replication errors. Such substances are ethidium bromide, used in laboratory for identification by fluorescence of nucleic acids separated through electrophoresis, and caffeine. After intercalating in DNA, other substances establish bridges between the polynucleotide strands through covalent bonds. Some reagents from this category are used in cancer therapy: mitomycin C and platinum salts (cisplatin). Psoralen is used in therapy of a skin disease named psoriasis. The psoralen is given per os (orally), is absorbed in intestine and is transported by the blood flow to the ill cells from skin. Exposure to ultraviolet light induces formation of bridges between the two chains of the DNA molecule and these mutations drive to the death of ill cells which are removed (desquamation) and the healthy skin remains. In Fanconi anemia (severe anemia, growth failure, severe immunodefficiency, frequent cancer of skin and liver) the reparatory mechanisms of lesions produced by substances that make bonds between chains are deficient. Physical mutagenic agents like the UV and ionizing radiations produce important lesions in DNA. The UV radiation, with wavelength around 300 nm, has energy per photon similar to a covalent bond. Because the nucleic acids strongly absorb the UV radiation, a specific lesion is produced in DNA: dimmerization of thymine, or of pyrimidines in generall. Two thymines from the same polynucleotide chain are linked through covalent links, and form a dimmer which distorts the duplex and arrests the replication. The cells have reparatory systems of lesions produced by UV, because these lesions are continuously produced (the Sun UV radiation is partially blocked by ozone layer from higher atmosphere, but some radiation reaches the Earth). These lesions are bigger than the point mutations and can be repaired in two ways. There is an enzyme, which uses light energy in order to disrupt the covalent link through a mechanism named photo-reactivation. The second mechanism is more complex and needs several enzymes. In the beginning, the polynucleotide chain is cut by an endonuclease, the zone with the dimmer is excised by an exonuclease, a correct chain is synthesized by a reparatory DNA polymerase and finally the DNA ligase links the new fragment to the old one. If there are deficiencies in the reparatory processes against UV, a skin genetic disease appear, named xeroderma pigmentosum. From the childhood on skin and cornea dry and atrophic zones appear which become malignant in time and the patients die with metastasis. The molecular defect with multiple variants consists in the impossibility of removal the zones with thymine dimmers. The ionizing radiation may be electro-magnetic radiations with very short wavelength (X or gamma rays), electrons ( radiation) or accelerated nuclei of helium ( rays). They have a common property: they remove electrons from atoms and this way ions and free radicals are formed.
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Lesions produced by ionizing radiations are complex: breaking of one or both DNA chains, base losses, changes of bases or of deoxyribose. The normal cells also have reparatory systems for these lesions. In ataxia teleangiectasia (failure of coordination of movements, dilatations of blood vessels visible like red spots on skin) the reparatory mechanisms of ionizing radiation-induced lesions is defective, but the reparation of lesions produced by UV is normal; there are severe immune deficiencies and decrease capability to destroy the malignant cells. Thus, the frequency of cancer is increased, especially of leukemia. The ionizing radiations are used in the radiotherapy of cancer, because the rapid proliferating cells are very sensitive to radiations; the reparatory processes of DNA need some time, and in case of rapid proliferation, a new replication cycle begins before the reparation is completed. Inhibitors of DNA biosynthesis are important in research for study of DNA replication mechanisms and in the medical practice, because they are used as antibiotics (anti-microbial or antiviral) and anti-cancer drugs. An antibiotic is a substance that in small doses inhibits bacteria multiplication through a selective action at the molecular level, and that doesnt affect the same process (if present) in the human organisms. Among the anti-microbial drugs which inhibit the DNA replication is the nalidixic acid. There are anti-viral reagents (one of the first used was phosphonoacetic acid) which selectively inhibits the viral DNA polymerases (enzymes encoded by the genetic material of viruses and synthesised in the host cell after infection). The phosphono-acetic acid acts only in very high concentrations on the DNA polymerases of the host cell. Among the anti-tumour substances that inhibit the DNA synthesis are arabinosyl-cytosine, arabinosyl adenine and neocarcinostatin, a protein made of 109 amino acids that disrupts the DNA molecule.