Binucleated cell

Watanabe R, Tanaka Y. 1982. Age-related alterations in the size of human hepatocytes A study of mononuclear and binucleate cells. Virchows Arch 39 9-20. 

HETEROKARYON A cell or fungal hypha that contains nuclei of more than one genetic origin nuclei in heterokaryons generally do not fuse, but can divide individually and simultaneously to form new multinucleate (or binucleate) cells or hyphae. 

Small lymphocytes from horses were transformed into larger radioresistant lymphoblast-hke cells following stimulation by phytohemagglutinin in vitro. One hundred rads x-irradiation killed all the small lymphocytes, but only around one-third of the lymphoblast-like cells (Dewey and Brannon, 1976). Horse lymphocytes were more radiosensitive than human lymphocytes. The ratios between doses inducing the same effect are 1.3,1.7, and 9.4 for the number of binucleated cells with micronuclei, micronucleus frequency in binucleated cells, and DNA synthesis inhibition, respectively (Catena et al, 1997). 

In the in vitro micronucleus test usually human peripheral blood lymphocytes are used. To distinguish cells that divided just once in culture, the cultures are treated with cytochalasin-B, a chemical that blocks actin polymerization and, as such, also cytokinesis. After one cell cycle, binucleated cells eventually with one or more micronuclei are obtained (Figure 8.7). It is necessary to distinguish the cells that divided in culture, because cell division is required for the formation of a micronucleus and a very small fraction of lymphocytes may have acquired micronuclei in vivo. Furthermore, micronuclei should be detected in cells that divided only once in culture in order to avoid an underestimation of the micronucleus frequency due to cell death. 

Figure 8.7 A cytochalasin-B-blocked binucleated cell, with two micronuclei.

Final cumulative doses of 22.2-29.6 GBq (600-800 mCi), with treatment intervals of 6-9 weeks, are reported in the literature. In this study, AR42J rat pancreatic tumour cells were grown in F12-K medium (GIBCO) supplemented with 10% foetal calf serum and incubated at 37°C. Cells (2.5 x Kf cells/mL) were cultivated on a 60 mm diameter plate and exposed to 2400 or 5700 kBq/ mL of [ LuJDOTATATE for 1 h, washed twice with PBS and then cultured with cytochalasin B (Sigma, USA) at a final concentration of 2 pg/mL for 72 h at 37°C. After 72 h, cells were fixed, stained and analysed for the presence of MN in binucleated cells. 

The cytogenetic data obtained showed that there was a positive correlation (p < 0.05) between the percentage of binucleated cells with MN (F)... 

FIG. 3.12. Dose-response curve for the percentage of binucleated cells (BNC) with micro-nuclei (MN) after 1 h of exposure of human peripheral lymphocytes to different radioactive concentrations of (a) p IJDOTATATE and (b) LuJDOTATATE. 

FIG. 3.14. Frequency of binuclealed cells (BNC) with micronuclei (MN) after 72 h culture of AR42J cells exposed to 2400 and5700 kBq/mL of I.u)DOTATATE. 

The results obtained showed the induction of MN in AR42J cells exposed to 2400 and 5700 kBq/mL of "l.u DOTATATE (Fig. 3.14). The frequency of binucleated cells with MN was 4.0% with exposure to 2400 kBq/mL of I Lu DOTATATE and 5.6% with exposure to 5700 kBq/mL of the radionuclide. Compared with basal values of 2.8% for binucleated cells with MN, there was a 1.4- and 2.0-fold increment, respectively. No alteration of the proliferation index was observed (PI = 1.23). 

For the in vitro micronnclens assay, a limit of abont 50% cytotoxicity is also appropriate. Moreover, for the in vitro micronnclens assay, since micronnclei are scored in the interphase snbseqnent to a mitotic division, it is important to verify that cells have progressed throngh the cell cycle. This can be done by nse of cyto-chalasin B to allow nuclear division bnt not cell division, so that micronuclei can be scored in binucleate cells (the preferred method for l)miphocytes). Other methods to demonstrate cell proliferation, including cell popnlation growth over time (PD)... 

Figure 11.7. Example of a binucleate cell with a micronucleus. See insert for color representation of this figure.

When cytokinesis is blocked, these fragments are visible as micronuclei in the resulting binucleate cells... 

With the aid of the binucleate cell technique (Kihlman, 1955a Gimenez-Martin et al. 1965), we have determined the mean duration of the mitotic cycle in A. proliferum to be 18-20 hr at 19°C. In autoradiographic experiments, a mean duration of 5 hr was found for the Gg period of interphase (Kihlman, unpublished). The satellite proved to be late replicating. 

When heterokaryons are formed between animal cells the nuclei in the same cytoplasm undergo synchronous initiation of DNA synthesis (Harris and Watkins, 1965) similar synchrony is observed in binucleate cells occurring in mouse embryo cultures (Church, 1967). DNA synthesis has been examined in multinucleate HeLa cells formed by fusion between cells in different phases of the life cycle (Rao and Johnson, 1970). There was a rapid induction of DNA synthesis in G1 phase nuclei following the fusion of G1 and S phase cells. When S phase cells were fused with G2 phase cells, no induction of DNA synthesis was observed in G2 phase nuclei and, no matter what the ratio of G2 S nuclei in the fused cell, the S phase nuclei continued DNA synthesis. 

Church, K. 1967. Pattern of DNA replication in binucleate cells occurring in mouse embryo cell cultures. Exp. Cell Res., 46 639-641. 

Since cell division is a prerequisite for MN expression, identification of mitosis is crucial. Until 1985, the method was hampered by the difficulty to identify the cells that divided in culture. Fenech and Morley introduced in 1985 the cytokinesis block in the methodology. The CBMN assay is based on the addition of cytochalasin-B, an actin inhibitor and therefore an inhibitor of cytokinesis, allowing the discrimination between cells that did not divide (mononudeated cells or MONO) and cells that divided once (binucleated cells or BN) or more (multinudeated cells) during in vitro culture. MN in mononudeated cells can represent the back ound frequency of MN (the frequency of MN that was present before... 

The most well-known purine alkaloid is caffeine (1,3,7-trimethylxanthine) 1 which, along with theobromine (3,7-dimethylxanthine) 2, accumulates in leaves of tea and mate and beans of coffee and cacao, which are popular components of nonalcoholic beverages and/or chocolate products [2]. The pharmacological effects of purine alkaloids in animals, such as stimulation of the central nervous system, have been investigated extensively [3]. Caffeine is also often utilized in cytological studies to induce the formation of binucleate cells and is, therefore, used to measure the duration of the mitotic cycle [4]. 

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