The cell cycle

The events of cell replication are embodied in the cell cycle, which has been divided into four successive time intervals G, S, G2, and D (or M) (Howard and Pelc, 1953) (Fig. 1). Gj is the time between the end of cell division and the start of DNA replication. S is the period of DNA replication, and G2 the time between the end of DNA replication and the start of mitosis. D (or M) is the period of cell division or mitosis. Go is postulated to be the state that a cell enters when cell replication is arrested at a critical point during Gi (Lajtha, 1963). Although the time periods for each state vary depending on species, cell type, and environmental conditions, approximate times can be given. Thus, Gi may last for 5 h, S for 7 h, G2 for 3 h, and D for 1 h, with a total generation time of 16 h. The Gj period is the most variable of the four periods (Sisken and Kinosita, 1961). 

At least two mammalian cells in tissue culture lack the Gi period Syrian hamster fibroblasts (Biirk, 1970) and the V79 line of Chinese 

A cytoplasmic factor is postulated to be responsible for the initiation of the S phase, and various investigators have demonstrated that DNA synthesis can be initiated by extracts of He La cells (Friedman and Mueller, 1968 Kumar and Friedman, 1972), L cells and ascites cells (Thompson and McCarthy, 1968), and embryos and eggs of Xenopus (Benbow and Ford, 1975). Although these factors are thought to be proteins, their exact nature and mechanisms of action are unknown. Recently it was suggested that diadenosine-tetra-phosphate (Ap4A) may act as an initiator of DNA replication in animal cells (Grummt, 1978). 

During the cell cycle, plasma membrane changes are observed, but 

Early in the S period, DNA has an average GC (guanine-cytosine) content of 43.6%, while in the late S period the GC content is 38.7%. This transition of synthesis from a relatively rich early GC DNA to a relatively rich late AT (adenine-thymine) DNA appears to be a general property of mammalian cells (Tobia et al., 1970), but its signficiance is unknown. 

The first evidence for a tumour-inducing virus was from sarcomas in domestic fowl. Rous discovered in 1907 that a cell-fi ee extract prepared from the minced extract of a sarcoma found in a Plymouth Rock caused sarcomas when injected into other domestic fowl. He postulated that the tumour was transmitted by a virus. The discovery received little attention then since it did not fit with the generally held theories of cancer at that time. Many years later, when a number of oncogenic viruses had been identified, the importance of Rous early discovery became apparent. Rous was awarded the Nobel prize 

This chapter begins with an outline of the cell cycle and the importance of normal transcriptional factors and proto-oncogenes, emphasising those characterised in avian species (almost entirely from the domestic fowl). The different classes of avian oncogene are then described, together with their relationship to proto-oncogenes. 

Several substrates of cyclin-dq endent kinase have been identified from in vitro studies, including nuclear lamins, vimentin, caldesmon, histone Hi and RNA polymerase 11 (Table 11.1) and these are the most likely physiological substrates. The onset of mitosis is marked by at least four morphological changes  

Substrate Possible role Phase of the cell cycle in which phosf orylated 

Nuclear lamins are intermediate filament proteins that polymerise to form the nuclear lamina, the structure underlying the nuclear membrane. They also form attachments to chromatin. In avian species, lamins have been classified on the basis of the amino acid sequence and their chemical properties into three groups A, Bi and Bj. The B lamins are 

CH 20 GROWTH AND DEATH OF CELLS AND HUMANS THE CELL CYCLE, APOPTOSIS AND NECROSIS 

Once the cycle has begun, the sequence of events is almost always completed in a time which is approximately constant for a given cell about 24 hours for a typical human cell. The largest variation in time occurs in the Gi phase. Very short cell cycles, 8 to 60 minutes, occur in early embryonic cells, during which cell division results in the formation of many smaller cells. In these cells, both the Gi and G2 phases are massively shortened, so that most of the time of cycling is spent in the S and M phases. 

A summary of some of these processes is as follows synthesis of phospholipids and cholesterol de novo synthesis of ribonucleotides synthesis of RNA de novo synthesis of deoxyribonucleotides regulation of synthesis of deoxyribonucleotides salvage pathways duplication of DNA transcription and translation (polypeptide synthesis). After this series of topics, those of fuels and ATP generation, mitosis and, finally, regulation of the cycle, are described and discussed. 

The pathways for the synthesis of phosphoglycerides and sphingolipids are described in detail in Chapter 11. They are, therefore, described only in brief here in order to emphasise the importance of the essential fatty acids in proliferation and how the cell cycle could be impaired by failure to provide these acids. 

The initial reactions produce phosphatidate, in which the two hydroxyl groups of glycerol 3-phosphate are esterified with long-chain fatty acids, catalysed by enzymes known as acyltransferases. An important point is that, due to the difference in specificities of the acyltransferase enzymes. 

An example of the time devoted to each phase of the cell cycle in the apical meristem of Vida faba roots is shown in Fig. 1, though these values vary somewhat in reports from different laboratories (MacLeod 1971). Cells which are not committed to nuclear division are sometimes viewed as entering a state Go (Gould et al. 1974, Peaud-Lenoel 1977), different from those shown in Fig. 1. Cells which begin differentiation leave the cell cycle and initiate a specialized pathway of development. 

While the linear sequence shown in Fig. 2.1 represents the simplest relationship of the phases of the cell cycle, an alternative model with more than one independent pathway between two successive steps has also been proposed (Peaud-Lenoel 1977). 

The nature of the signals controlling the cell cycle or initiating differentiation is unknown. Possibilities include physical factors, e.g., position relative nucle-ar/cytoplasmic volume, etc. (Roberts 1976), genetic factors, e.g., de novo synthesis or activation of specific enzymes (Van t Hof 1974, Mitchison and Creanor 1969) and chemical factors, e.g., hormones (Nagl 1972). 

Growing cells are characterised by a sequence or sequences of events leading to duplication of their constitutents. These events appear to occur in a strict, temporal order, and growing cells may be considered as a simple system for the study of gene expression. The two most obvious events which occur in growing cells are cell division and DNA synthesis which are the markers used to characterise the cell cycle (Fig. 10.1). 

M or mitosis is the period when the cells divide and S is the period of DNA synthesis while G1 and G2 represent gaps — gaps in our knowledge of obvious markers in these areas. Much cell biology in recent years has been devoted to attempts to fill these gaps. These 

Mitosis is heralded by the rounding up of the cell, and the first visible indication that it is about to divide is a change in appearance 

The centrioles migrate to opposite poles of the cell and the mitotic spindle is formed, apparently joining the cell membrane through the centrioles to the centromere of each chromosome. Spindle fibres consist of one type of protein, tubulin, of molecular weight 60,000. It is the organisation of these molecules to form the mitotic spindle which is blocked by the drugs colchicine, colcemide, nocodazole, vincristine and vinblastine (Fig. 10.3) with the consequence that mitosis is arrested in metaphase. 

At anaphase the two sets of chromosomes move to opposite poles due to tubulin action, and by telophase the chromosomes decon-dense as the cell membrane encloses each new daughter cell. The paired cells are still rounded up and resemble a dumbell, but very soon they flatten out as the nuclear membrane and nucleoli reform and the cells enter Gl. 

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Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Dacarbazine is activated by photodecomposition (chemical breakdown caused by radiant energy) and by enzymatic N-demethylation. Formation of a methyl carbonium ion results in methylation of DNA and RNA and inhibition of nucleic acid and protein synthesis. Cells in all phases of the cell cycle are susceptible to dacarbazine. The drug is not appreciably protein bound, and it does not enter the central nervous system. 

Antimetabolites interfere with normal metabolic pathways. They can be grouped into folate antagonists and analogues of purine or pyrimidine bases. Their action is limited to the S-phase of the cell cycle and therefore they target a smaller fraction of cells as compared with alkylating agents. 

In general, the mechanisms of action are not cell cycle specific, although some members of the class show greatest activity at certain phases of the cell cycle, such as S-phase (anthracyclins, mitoxantrone), Gl- and early S-phases (mitomycin C) and G2- and M-phases (bleomycins). 

The eukaryotic somatic cell cycle is defined by a sequential order of tasks a dividing cell has to complete it must replicate its DNA, segregate its chromosomes, grow, and divide. The cell cycle can be divided into four discrete phases. DNA replication is restricted to S phase (DNA synthesis phase), which is preceded by a gap phase called G1 and followed by a gap phase called G2. During mitosis (M phase) the sister chromatids are segregated into two new daughter nuclei and mitosis is completed by the division of the cytoplasm termed cytokinesis (Fig. 1). 

DNA synthesis during S phase of the cell cycle resulting in a doubling of the genomic DNA. Replication can be subdivided into three distinct phases initiation, elongation, and termination. 

The phase of the cell cycle where the sister chromatids are separated and distributed onto two daughter nuclei. First, upon entry into mitosis the chromosomes are condensed followed by the breakdown of the nuclear-envelope (prophase). The two centrosomes are separated and induce the formation of the mitotic spindle. Then, the chromosomes are captures by the spindle and aligned on the metaphase plate (metaphase). The sister-chromatids are separated and pulled to the poles of the spindle (anaphase). In telophase, two new nuclei are formed around the separated chromatids. 

The phenanthroindolizidine alkaloid (-)-antofine (95) exhibits high cytotoxicity to drug-sensitive and multidrug-resistant cancer cells by arresting the G2/M phase of the cell cycle. In the first asymmetric total synthesis of (-)-95, the late-stage construction of pyrrolidine 94 for the final Pictet-Spengler cyclo-methylenation to 95 was performed by RCM and subsequent hydrogenation (Scheme 18) [67]. 

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