DNA synthesis arrest

Fig. 20, Defective phenotypes of conditional mutants of T4D under restrictive conditions. Characterized genes are represented by shaded areas illustrating relative locations, and, if known, approximate map lengths. The enclosed symbols indicate defective phenotypes as follows DNA NEG. noDNA synthesis DNA ARREST DNA synthesis arrested after a short time DNA DELAY DNA synthesis commences after some delay MAT DEF. maturation defective, DNA synthesis is normal but late functions are not expressed a hexagtm indicates that free heads are produced, an inverted T, that free tails are produced TAIL FIBER fiberless particles produced gene 9 mutants produce inactive particles with contracted sheaths g e 11 and 12 mutants produce ftagile particles which dissociate to free heads and free tails. (Edgar and Wood, 1966).

In view of the well-documented inhibition of dihydrofolate reductase by aminopterin (325), methotrexate (326) and related compounds it is generally accepted that this inhibitory effect constitutes the primary metabolic action of folate analogues and results in a block in the conversion of folate and dihydrofolate (DHF) to THF and its derivatives. As a consequence of this block, tissues become deficient in the THF derivatives, and this deficiency has many consequences similar to those resulting from nutritional folate deficiency. The crucial effect, however, is a depression of thymidylate synthesis with a consequent failure in DNA synthesis and arrest of cell division that has lethal results in rapidly proliferating tissues such as intestinal mucosa and bone marrow (B-69MI21604, B-69MI21605). 

BrdU analysis allows to distinguish and quantify if there is an arrest in DNA synthesis, in which compartment of S phase there are and it is possible to separate E (early) S from Gi or L (Late) S from G2/M. This advantage is demonstrated in Figure 5, where HCT-116 cells were treated with SN-38 or edotecarin that affect DNA replication [21],... 

Indeed, TCA (42) at a concentration of 10 Xg/mL, has been shown to elevate levels of ROS, as measured by flow cytometry. Consistent with earlier observations regarding structure-activity relationships, Me-TCA (44) showed 3-fold induction of ROS while dihydro-TCA (43) had no effect on the cellular levels of ROS.It is noteworthy that parthenolide (45), a sesquiterpene natural product structurally related to TCA, has previously been shown to increase the levels of ROS by glutathione depletion in hepatocellular carcinoma cell lines. In a separate study, parthenolide was able to inhibit DNA synthesis, cause cell cycle arrest, and induce apoptosis which are important mechanisms for controlling tumor growth. 

Several effects of forskolin on B-lymphocytes, the cells of the immune system responsible for the production of immunoglobulins, have further been reported. This diterpene was found to inhibit cellular proliferation of B cells stimulated either by antibodies to surface immunoglobulins (anti-mu), and an antibody to CD20 antigen or 12-O-tetradecanoyl phorbol 13-acetate [219]. There was also a clear inhibition of G1 entry and DNA synthesis, and forskolin maintained its inhibitory effect even when added later after anti-mu stimulation. Additionally, no differences were found in the inhibitory effect of forskolin on neoplastic B cells, as compared to the responses of normal cells. Growth inhibition associated with an accumulation of cells in G1 was later found when cells of the B-lymphoid precursor cell line Reh were incubated with forskolin [220]. In that study, a delay of cells in G2/M prior to G1 arrest was observed, suggesting that important restriction points located in the G1 and G2 phases of the cell cycle may be controlled by forskolin (due to cAMP levels elevation). In a subsequent study [221], it was found that the arrest of Reh cells was accompanied by rapid dephosphorylation of retinoblastoma protein, which was suggested to be a prerequisite for the forskolin mediated arrest of these cells in Gl. 

A 34 kDa protein (p34) plays an important function in the control of the cell cycle in all eukaryotes. It was first identified as the product of the cdc 2/cdc 28 gene in yeast mutants which caused cells to be arrested at a commitment point in G1 (Murray, 1981). However, anti-p34 antibodies injected into cells do not affect DNA synthesis but block cells in mitosis (Riabowol et al., 1989) and p34 is believed to function both at the onset of S-phase and at mitosis. 

Temin (1970) and Todaro et al. (1965) showed similar effects for chicken fibroblasts and 3T3 mouse fibroblasts. The low level of serum is important for survival as well as for the subsequent stimulation of DNA synthesis (Cherrington, 1984). A kinetic analysis using time lapse cinematography (Zetterberg and Larsson, 1985) showed that Swiss 3T3 cells were only susceptible to cell cycle arrest in a short period (3-4 h) following mitosis. Even a 1-h exposure to serum-free medium during this time forced the cells into GO from which they required 8 h to return to Gl. The length of the postmitotic sensitive phase was very constant at between 3 and 4 h but considerable intercellular variability existed in the duration of the pre S-phase Gl period consistent with a transition probability event ( 10.4). 

Although most cultured cell populations come to rest in Gl, i.e. between cell division and DNA synthesis, a proportion of mouse ear epidermal cells are thought to be arrested in G2 (Gelfant, 1959, 1963), although recent evidence casts doubt on these conclusions (Sauerbom et al., 1978). There is some evidence that human embryonic fibroblasts maintained in culture for 48 passages (i.e. in the terminal phase) may arrest in G2 (Maciero-Coelho et al., 1966) but these are so abnormal as to be of no value in studies of G2. As mentioned in 11.6, some tumour cells arrest in G2 on medium exhaustion, but again many metabolic processes are affected and the cells cannot be considered as typical of G2-phase cells. 

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