Colcemid

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. 

Borsuk E, Manka R 1988 Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid. Gamete Res 20 365-376 Ciemerych MA 1995 Chromatin condensation activity and cortical activity during the first three cell cycles of mouse embryo. Mol Reprod Dev 41 416—424 Ciemerych MA, Kubiak JZ 1999 Transient reactivation of CSF in parthenogenetic one-cell mouse embryos. Biol Cell 91 641—647... 

The chromatid separation process has also remained mysterious. It is an autonomous process that does not direcdy depend on the mitotic spindle (Wilson 1925, Mazia 1961). This is most vividly seen in cells whose spindles have been destroyed by spindle poisons such as colchicine. In many organisms, in particular in plant cells, the cell cycle delay induced by colchicine is only transient and chromatids eventually split apart in the complete absence of a mitotic spindle (Mole-Bajer 1958, Rieder Palazzo 1992) (Fig. 2). Mitosis in the presence of colchicine or colcemid (known as c-mitosis) leads to the production of daughter cells with twice the normal complement of chromosomes. This process is routinely used for manipulating plant genomes and may contribute to the therapeutic effects of taxol in treating breast cancer. 

Rieder CL, Palazzo RE 1992 Colcemid and the mitotic cycle. J Cell Sci 102 387-392 Saez C, Japon MA, Ramos-Morales F et al 1999 hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias. Oncogene 18 5473—5476... 

Observations are made in metaphase cells arrested with a spindle inhibitor such as colchicine or colcemid to accumulate cells in a metaphase-like stage of mitosis (c-metaphase) before hypotonic treatment to enlarge cells and fixation with alcohol-acetic acid solution. Cells are then dispersed on to microscope slides and stained and slides are randomized, coded and analyzed for chromosome aberrations with high-power light microscopy. Details of the procedure are given in Dean and Danford (1984) and Preston et al. (1981, 1987). The UKEMS guidelines (Scott et al., 1990) recommend that all tests be repeated regardless of the outcome of the first test and... 

Metaphase Analysis. Metaphase analysis can be performed in any tissue with actively dividing cells, but bone marrow is the tissue most often examined. Cells are treated with a test compound and are arrested in metaphase by the administration of colcemid or colchicine at various sampling times after treatment. Preparations are examined for structural chromosome damage. Because the bone marrow has a good blood supply, the cells should be exposed to the test compound or its metabolites in the peripheral blood supply. Additionally, these cells are sensitive to S-dependent and S-independent mutagens (Topham et al., 1983). 

For monolayer cultures, the cultures are set up the day before BrdU treatment so that the cells will be in exponential growth before the addition of BrdU or the test compound. After BrdU addition the cells are allowed to undergo the equivalent of two cell cycles before cell harvest. A spindle inhibitor such as colchicine or colcemid is introduced for the final 1-2 h of culture to arrest cells in metaphase, after which the cells are harvested and chromosome preparations are made by routine cytogenetic techniques. 

The role of microtubules in neutrophil function can be investigated using agents such as colchicine, colcemid, vinblastine and vincristine, which disrupt these structures. Stimulation of neutrophils with chemotactic agents causes a rapid and transient assembly of microtubules, but this assembly does not affect chemotaxis. Similarly, cytoplasts (neutrophils devoid of nu-... 

The protein content is measured by biochemical methods, such as methods of Lowry or Bradford [14,18,19,21,23], now usually by commercially available kits. The DNA content can be measured by flow cytometry, and the chromosomes can be counted directly in cells after their arrest in the metaphase of the mitosis by Colcemide [21]. 

Phytohemagglutmin (PHA) (Gibco-BRL, Gaithersburg, MD) reconstitute as directed and add sufficient quantity to Ham s F-10 medium (usually about 1-2 mL/100 mL). Colcemid stock solution (Gibco-BRL) 10 ag/mL in phosphate-buffered saline (PBS). Miscellaneous glass and plasticware, such as pipets, centrifuge tubes, microscope slides, and number-one coverslips. 

One hour before harvest, add sufficient colcemid stock to provide a final concentration of 0.8 pg/mL. Incubate. 

After treatment, the exposure mixture was removed and cells were grown for 18 h at 37 °C in medium plus serum. Then, 1 /zg of colcemid (Gibco) was added per tissue culture flask, and cells were incubated for another 2 h at 37 °C. The collection of the cells by trypsinization, their fixation, and the staining of chromosomes with Ciemsa were carried out according to standard procedures (18). At least 50 metaphases were screened for chromosomal aberrations per treatment group. 

Colcemid Dissolve 5 pg/mL of distilled water. Filter-sterilize, and store in aliquots at —20°C. 

As far as cancer is concerned, disturbances of microtubular organization do not appear to play an impotrant role (10, 11,12). Disturbances of these structures by colcemid and loss of long spiky pseudopodia do not affect the capacity of cancer cells to invade normal tissues. 

The formation of the neurite-like processes appears to be dependent on assembly of microtubules as colchicine and Colcemid, antimicrotubule drugs, prevented shape changes in the presence of butyrate (2,8). The amount of tubulin per cell did not change when HeLa were treated with butyrate (R.C.Henneberry, unpublished observations). The role of microtubule assembly was further explored with a calcium ionophore which alters intracellular calcium levels and thus promotes microtubule depolymerization. 

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). 

COLCEMID A chemical that inhibits the formation of the mitotic spindle that is involved in the division of eukaryotic cell nuclei colcemid (or the related substance colchicine) is used in some procedures to facilitate cytogenetic analysis. 

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. 

A much simpler, though in general less satisfactory procedure, is to grow cells on coverslips and treat as above with colcemid. Treat the coverslip for 30 min with warm, hypotonic saline (see c above) and then fix for 10 min and dry at room temperature. Stain the cells and mount the coverslips, cells downwards. The difficulty with this method is that cells are easily dislodged from the substratum on adding the fixative. For this reason this step should take at least 10 min. 

Growing cells are treated with colcemid (0.4 /ig/ml) for 3 h at 37°C and if they are growing as a monolayer, they are then harvested by trypsinisation. 

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. 

Fig. 10.9. Accumulation of mitotic cells. The mitotic collection function (log 1 + Nm) is plotted against time for Chinese hamster cells (T = 12.4 h) and HeLa S3 cells ( T — 20.1 h) to which colcemid was added at zero time. (Reproduced from Puck, 1964, with kind permission of the author and publisher.)...

If tritiated thymidine is added along with colcemid three accumulation functions can be measured (a) total labelled cells, (b) total mitoses, (c) labelled mitoses, which enables calculation of the length of S and G2 and hence also Gl. 

Fig. 10.10. Accumulation of labelled mitotic cells. A similar experiment with HeLa S3 cells to that shown in Fig. 10.9 except that [3H]thymidine was added with the colcemid at zero time. After an initial lag the lines for accumulation of all mitotic cells and labelled mitotic cells are parallel and separated by tG2. (Reproduced from Puck and Steffen, 1963, with kind permission of the authors and publisher.)...

All this information may be obtained in an experiment lasting 3-4 h with or without the use of colcemid. The graph (Fig. 10.12) is then plotted on single cycle semi-log paper as follows ... 

Fig. 10.12. Graphical analysis of cell cycle. 5 cm Petri dish cultures of Aedes albopictus mosquito cells were set up and the number of cells per dish counted regularly to establish the doubling time (taken as the generation time). When it was clear that the cells were growing exponentially [3H]thymidine and colcemid were added and cells processed to establish % cells labelled, mitotic index and tG2. The graph was drawn as described in the text (see 10.7.4.)...

After 20-30 min the cells divide synchronously. Again this method is not universally applicable as not all cell lines show reversible colcemid inhibition. 

Cells in mitosis may be selectively removed and discarded over a long period of time. This leaves behind a population most of which should be in G2-phase (Stubblefield, 1964 Creasey and Markiw, 1965 Pfeiffer and Tolmach, 1967). Unfortunately, as well as the probability of selecting slow growers, the use of colchicine, colcemid and vinblastine may not only inhibit mitosis but may also have other effects on cells such as inhibition of RNA synthesis. 

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. 

Colcemid included in the medium the last three hours... 

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