Colcemid block

Several groups of drugs that bind to tubulin at different sites interfere with its polymerization into microtubules. These drugs are of experimental and clinical importance (Bershadsky and Vasiliev, 1988). For example, colchicine, an alkaloid derived from the meadow saffron plant Colchicum autumnale or Colchicum speciosum), is the oldest and most widely studied of these drugs. It forms a molecular complex with tubulin in the cytosol pool and prevents its polymerization into microtubules. Other substances such as colcemid, podophyllotoxin, and noco-dazole bind to the tubulin molecule at the same site as colchicine and produce a similar effect, albeit with some kinetic differences. Mature ciliary microtubules are resistant to colchicine, whereas those of the mitotic spindle are very sensitive. Colchicine and colcemid block cell division in metaphase and are widely used in cytogenetic studies of cultured cells to enhance the yield of metaphase plate chromosomes. 

Butyrate appears to induce sialyl transferase activity as addition of actinomycin D or cycloheximide to the medium along with butyrate blocked the increase in activity (4,8). Specific cell cycle inhibitors such as thymidine and colcemid did not cause an increase in activity in control cells or prevent induction in butyrate-treated cells (8). Induction of sialyl transferase activity also occurred in serum-free medium (8). When homogenates of control and butyrate-treated cells were admixed and assayed for sialyl transferase activity, there was no evidence of an inhibitor in the former or activator in the latter cells (8). 

As even in an exponential culture only a small proportion of cells are in mitosis, it is necessary to increase this number by addition of colcemid or nocodazole which block cells in mitosis ( 10.2). This also has the effect of separating individual chromosomes by its action on the spindle. 

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. 

A certain amount of information can be gained by measuring the accumulation of mitotic cells on addition of a mitotic blocking agent. Cultures may be set up as before and colcemid (0.25 /xg/ml) added at zero time. 

This unbalanced growth rapidly leads to cell death if prolonged for more than a generation time (Ruekert and Mueller, 1960). Unbalanced growth will occur in any cells committed to division ( 10.4) yet blocked in one function including those maintained in colcemid for more than a few hours. Moreover, selective blocking of DNA synthesis may have effects which are not apparent until the synchronised cells are released and proceed to the next G1-phase (Firket and Mahieu, 1966 Cress and Gerner, 1977). Schindler et al. 

The selection of mitotic cells has been described in 11.2 and the proportion of cells in mitosis can be increased by use of the mitotic blocking agents colcemid or preferably nocodazole. The combination of a single thymidine block for 15-16 h followed by a 4-5 h in 0.04/ig/ml nocodazole gives a yield of 25-30% when mitotic cells are harvested (Zieve et al., 1980) and further mitotic cells can be obtained with subsequent shakings. 

Colchicine and a synthetic relative, colcemid, have long been used as mitotic inhibitors. In cells exposed to high concentrations of colcemid, cytosolic microtubules depolymer-ize, leaving an MTOC. However, when plant or animal cells are exposed to low concentrations of colcemid, the microtubules remain and the cells become blocked at meta-... 

Method Details CELL DIVISION WAS BLOCKED BY COLCEMID 0. 2 UG/L STAINED WITH 1. 3 % GIEMSA... 

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