Analysis of Preparations and Data Intepretation

As in every procedure of interphase molecular cytogenetics, labeling must be accurate to minimize the occurrence of false-positive as well as false-negative results. For example, a high frequency of centromere-negative centromeres would indicate that the agent inducing them acts by chromosomal breakage; a poor labeling would produce false-negative results, leading to the wrong conclusions about the mechanism of action of the agent under study. After application of the protocols described here, the quality of labeling can be immediately appreciated using the fluorescence microscope. The performance of the reaction must be considered acceptable only if nuclei are evenly labeled in the working area. If fluorescent spots are faint or if labeling is uneven along the slide, the micronucleus assay cannot be conducted because the chance of false-negative results is too great. The performance of the PRINS technique is on the average high, and repeated experiments giving poor quality labeling must prompt one to analyze step by step the possible reasons of failure.

3.4.1. Selection of Cells To Be Analyzed

The scoring of acceptable slides must proceed along the working area of the slide, at X1000 magnification; the minimum number of cells to be analyzed per replicated experimental point is 1000. However, refer to the general protocols for micronucleus assay for the recommended sample size (3,4,6). Cells must be discarded from analysis if:

a. The fluorescent spots corresponding to the sequence(s) of interest are faint.

b. The nucleus does not have a regular shape with definite boundaries.

c. Different cells are overlapping in the same area.

d. A micronucleus is found but is not clearly contained in the cytoplasm of the reference cell (see Note 22).

e. A micronucleus is found but is not clearly separate from the main nucleus (it could derive from nucleus extrusion).

f. The micronucleus and the nucleus are not on the same focus plane.

g. The diameter of the micronucleus exceeds one-third of that of the main nucleus of the cell (again the origin of the body of extra-chromatin could be different from that postulated for the micronucleus assay).

h. The shape of the micronucleus is not regular: micronuclei have a proper membrane, therefore accept only round or oval micronuclei with a clear boundary.

i. Micronucleus and nucleus stainings are of different intensities.

j. The micronucleus staining is refractile.

k. Multiple micronuclei are found: accept only cells with not more than two micro-nuclei to avoid including apoptotic cells in your analysis.

3.4.2. Classification of Micronuclei

The cells selected for analysis are classified, if carrying a micronucleus, with respect to the presence or absence of sequences labeled.

1. If a single-color PRINS is conducted, micronuclei can be classified as "positive" when the fluorescent spot is observed. For example, C+MN if the micronucleus contains the centromeric fluorescent spot (see Notes 23 and 24).

2. The number of fluorescent spots visible in each micronucleus must be recorded when telomere sequences are under analysis because the number of telomeric regions expected depends on the modality of formation of the micronucleus itself (Fig. 2C; see Notes 23 and 25).

3. If a dual-color PRINS procedure is conducted, the classification of micronuclei must proceed step by step in order to minimize bias classifications (see Note 26).

3.4.3. Quantitative Analysis and Discussion of Data

After microscope analysis, calculate (1) the frequency of cells carrying micronuclei with respect to the total number of cells scored. In a control cell population, expected values are in the order of 0.1-0.5%; in treated population these values may be up to ten times higher. (2) The frequency of micronucleated cells belonging to every category observed (for example: C+MN vs C-MN), with respect to the total number of the cells analyzed.

Use the frequencies to infer the main mechanisms of action of the agents under study (apply the appropriate statistical comparisons): agents acting with a prevalent clastogenic mechanism will produce a large number of centromere-negative micronuclei; on the contrary, aneuploidogenic agents will increase specifically the frequency of centromere-positive micronuclei. In the application of the tandem labeling approach, you can, in addition, consider the occurrence of preferential events of breakage into the pericentromeric heterochromatic region by calculating the proportion of micronuclei that are positive for the major satellite sequence on the total number of micronuclei deriving from chromosome breakage (i.e., those positive for major satellite plus negative ones). If the proportion will exceed that expected on the basis of the size of major satellite DNA (5-10%), a preferential clastogenic action on the sequence investigated must be postulated. In the dual-color labeling of centromeric and telomeric sequences, you will compare the relative frequencies of centromere-positive or -negative micronuclei to detect aneuploidogenic vs clastogenic action, and the number of telomeres will allow one to understand whether segregation errors occur more often before or during anaphase (Fig. 2C). Note finally that, according to the sequence(s) selected for analysis, some classes of micronuclei are not expected unless false-positive or false-negative results occurred (see Note 27).

4. Notes

1. The cytocentrifuge is particularly useful when the number of cells available is small, or for those cell types that are sensitive to the fixative treatment. Limited volumes of living cell suspensions can be sufficient to obtain perfectly flat cell preparations, which will be postfixed easily.

2. This solution is not stable and quickly loses its fixative properties.

3. The quality of slide surface is crucial for successful cell preparations and for the performance of PRINS reactions. For the best reproducibility, choose only high-quality, thin microscope slides. Although the slides are provided precleaned, it is recommended that they are washed immediately before use in detergent solution (in a hot temperature for 15-30 min). Slides are then rinsed in tap water and finally three times in distilled water. This step will be of help in keeping the background fluorescent noise as low as possible. Clean slides should be used immediately. Drop cell preparations preferentially on wet slide surfaces.

4. Different companies provide thermocyclers equipped with flat blocks, which are specifically designed for in situ PCR applications. A cheap alternative is the use of two thermoblocks, respectively, set at the annealing and extension temperatures. In both situations, the reproducibility of the protocol depends on how accurate the thermal profile is around the employed surface, and along the reaction time.

5. For single labeling, we recommend the use of digoxigenin-11-dUTP detected by fluoresceinated antibodies because, at least in our hands, this labeling system appears as the most efficient. Alternatively, a different modified nucleotide (e.g., biotin-16-dUTP) at the same final concentration indicated for digoxigenin-11-dUTP can be used, together with the appropriate detection protocol, as that described in Subheading 3.3.

6. This enzyme is strongly recommended because it gives highly reproducible results and produces sharp fluorescent spots.

7. The nail polish must be resistant to temperature because a possible pitfall is the low quality of sealing. Temperature-sensitive nail polish tends to bubble. Consequently, mix evaporation can occur, leading to failure of the reaction.

8. The micronucleus assay can be conducted with several primary cells or cell lines. The major applications of the micronucleus assay are on human, murine, or hamster cells. The only crucial requirement concerns the use of proliferating cells because the micronucleus originates at cell division. It must be reminded that the micronucleus is the ultimate consequence of a primary event which may have been occurred in a previous phase of the cell cycle. For example, it is well known that chemically-induced chromosome breakages are restricted for a large number of agents to the Gj phase. Therefore, the experimental planning must take into account the specific cell cycle timing for the cells of interest, either in vivo or in vitro.

9. Standard trypsin-EDTA should be used for adherently growing cell lines or the recommended enzymes for dissecting cells from different tissues. Whichever protocol is used, remember that prolonged exposure to proteolytic enzymes must be avoided because it can damage cell membranes, leading to low-quality slide preparations.

10. Few trials will be sufficient to gain the experience concerning the optimal cell density required in these experiments. Analysis of low-density preparations is both time-consuming and expensive. In contrast, high-density preparations lead to extensive cell overlapping and, moreover, make it difficult to control denatur-

ation conditions along the slide area. Only Shandon Cytospin provides high-quality features for cell spotting on slides by cytocentrifugation. The Cytospin Shandon has a unique rotor and speed is only given in rpm by this manufacturer.

11. Avoid the use of PBS because the salt will deposit on the slide surface during air-drying, impairing the immediate observation of the preparation at the contrast phase microscope. If you have many cell samples to be managed at the same time or if you must work with high cell numbers, it is preferred to resuspend the cells at a higher cell density than indicated and to prepare serial dilutions immediately before use. In this way the cell integrity will remain well preserved.

12. If only a fine adjustment of the cell density is necessary, you can simply modify the volume to be spotted per slide. However, remember that cell aggregates may be formed in small volumes, whereas high volumes result in a less tight adhesion of the cells to the slide surface.

13. Some cell types are sensitive to air-drying after cytocentrifugation, which results in poor quality cytoplasm and nucleus morphology. If irregular boundaries or vacuoles are observed, it could be better to immerse the slides as soon as possible in ethanol to avoid air-drying. In this case, check for slide quality under the contrast phase microscope by keeping the slides perfectly wet.

14. According to the experimental rationale, single-color PRINS can be performed by using one of the probes listed in Table 2.

15. The denaturation step can be controlled more easily by handling few slides per time. The denaturation time depends on the cell type used. The time indicated is a good starting point to establish the optimal conditions. You can check under the contrast phase microscope the quality of the cells after the denaturation has been completed: denatured cells appear pale, but maintain their morphology. After PRINS, cells that are inadequately denatured will show only small fluorescent signals, and the labeling efficiency will be, on average, low (see Notes 24 and 25); the shape of fluorescent signals will appear diffused, and the nucleus morphology will be in part lost, if the denaturation was too effective. In addition, in the presence of strong denaturation conditions, small micronuclei can be completely lost. All these aspects can influence the accuracy of analysis. Remember finally that the cell density is crucial, and you will hardly obtain proper denatur-ation with high-density preparations.

16. For some cell types, we have found that heat denaturation can be more efficient than NaOH denaturation. For example, the highly compact chromatin of mouse spermatids and spermatozoa can be denatured at 94 to 96°C for 5 min (place 400 mL of 10 mM Tris-HCl, pH 7.6, on the slide and cover it with a 24 x 40-mm cover slip. Rinse for 2 min in Tris-HCl on ice). Also, depending on the preparation, a proteolytic treatment can be applied before PRINS, but it does not appear crucial for the majority of cell types.

17. To optimize the process, let a new slide drain while spotting the reaction mix on the previous one. Remember that slides must never dry completely.

18. The humidified chamber is a plastic box in which a layer of wet paper in the bottom prevents slides from evaporating, and cover slip sealing is not required.

19. If the cover slip remains tightly adhered to the slide, it is possible that evaporation of the reaction mix occurred, and in this case the labeling could be unsuccessful. Check for the quality of nail polish sealing (see Note 7).

20. For the best results, we suggest to label in the first cycle the sequence expected to do the smallest fluorescent spot. Therefore, label minor satellite DNA before major satellite, or telomeric sequences before minor satellite. Also, use the most sensitive detection system (digoxigenin/anti-digoxigenin) for the first PRINS cycle.

21. When applying a dual-color procedure and, more importantly, a tandem labeling procedure, it is crucial to avoid that the previous mix components remains available during the second PRINS reaction. A washing step after the stop solution is strongly recommended.

22. Contrast phase combined with ultraviolet light allows the accurate localization of micronuclei. However, the cytoplasmic boundaries can be detectable simply because the very faint background fluorescence.

23. Spots corresponding to either centromeric or telomeric sequences are preferentially located at the micronucleus boundary, but occasionally can be observed in inner positions. If fluorescent spots with intensity comparable to that observed into the micronuclei are visible in the cytoplasm, discard the cell from the analysis to avoid false-positives.

24. A positive control, i.e., a chemical with known aneuploidogenic activity can be used to check whether the frequency of centromere positive micronuclei is maximized as expected (Fig. 2A). If the frequency of centromere lacking micronuclei is still high, a false-negative result was probably obtained. Improve denaturing conditions as a possible cause for the underestimation of fluorescent signals. Micronuclei with more than one fluorescent spot are seldom observed. Record the occurrence of this event separately.

25. At least one fluorescent spot is expected in the micronuclei (Fig. 2C). Therefore, negative micronuclei must be considered artifacts and their frequency should be close to zero. Improve denaturing conditions as a possible cause for the underestimation of fluorescent signals.

26. Always score the fluorescent spots in the same order considered for labeling: minor satellite before major satellite sequences, telomeric sequences before minor satellite. The bias classification of micronuclei is lowest by choosing this order. Fluorescent spots corresponding to centromeric and pericentromeric signals must be juxtaposed (use the triple band filter to verify the tandem labeling). The observation of separated sequences must be recorded separately.

27. For example, you never expect micronuclei completely lacking telomeric sequences, or micronuclei carrying the minor satellite but lacking the major one. Micronuclei carrying the centromere should show at least two telomeres because they are expected to harbor a whole chromosome (Fig. 2C), even though a limit situation would be a micronucleus originating from the missegregation of a chromosome carrying a terminal deletion.

References

1. Heddle, J. A. (1973) A rapid in vivo test for chromosome damage. Mutation Res. 18, 187-193.

2. Schmid, W. (1975) The micronucleus test. Mutation Res. 31, 9-15.

3. Krishna, G. and Hayashi M. (2000) In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutation Res. 455, 155-166.

4. Fenech, M. (2000) The in vitro micronucleus technique. Mutat. Res. 445, 81-95.

5. Russo, A. (2002) PRINS tandem labeling of satellite DNA in the study of chromosome damage. Am. J. Med. Genet. 107, 99-104.

6. Russo, A. (2000) In vivo cytogenetics: mammalian germ cells. Mutat. Res. 445, 167-189.

7. Masumoto, H., Masukata, H., Muro, Y., Nozaki, N., and Okazaki. T. (1989) A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J. Cell Biol. 109, 1963-1973.

8. Bertoni L., Attolini, C., Faravelli, M., Simi, S., and Giulotto, E. (1996) Intrachromosomal telomere-like DNA sequences in Chinese hamster. Mamm. Genome 7, 853-855.

9. Mitchell, A., Nicol, L., Mallory, P., and Gosden, J. (1993) Novel structural organization of a Mus Musculus DBA/2 chromosome shows a fixed position for the centromere. J. Cell Sci. 106, 79-85.

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