[20 Methods for Chromosome Banding of Human and Experimental Tumors in Vitro

By Jeffrey M. Trent and Floyd H. Thompson

Chromosomal analysis is increasingly becoming a necessity in studies of somatic cell genetics. The major emphasis of this chapter will be to describe several common cytogenetic procedures which can be utilized to identify the chromosomal constitution of human and experimental tumors grown in vitro. Emphasis has been placed on discussion of methods for banding analysis of established cell lines, although a brief description of procedures for analysis of primary tumor material has also been included. Additionally, the discussion has been principally centered around banding analysis of human tumor cells in culture, although the techniques described are amenable to normal and abnormal cells from other mammalian species.

Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 151 All rights of reproduction in any form reserved.

Chromosome Characterization: General Comments

The following procedures are commonly used for chromosomal characterization of mammalian cells grown in vitro. Additional procedures for chromosome banding may be found in texts devoted solely to methodology.1,2 Investigators who are considering using cytogenetic techniques in order to identify cell line cross-culture contamination are referred to the excellent recent article of Pathak and Hsu3 which details procedure and criteria for identification of interspecies cell line contamination.

In order to fully characterize the chromosomal complement of a cell line or primary tumor specimen, it is necessary to employ one or more of several "chromosome banding" procedures. The most informative and commonly used banding techniques include G-, Q-, or R-banding (described below); all are procedures which induce a pattern of longitudinal differentiation (banding) along the length of each individual chromosome. With sufficient experience in band pattern recognition, it is possible to unequivocally identify all individual normal chromosomes. However, inevitably in cell lines derived from tumors as well as nontransformed cells grown in long-term culture, numeric and structural chromosome alterations will be found. Some of these changes result in the production of "marker" chromosomes, chromosomes which have undergone structural rearrangement to a point where they cannot be fully identified by G-, Q-, or R-banding. In these instances, use of techniques which stain a restricted number of specific bands or structures may be of benefit [e.g., banding procedures for constitutive heterochromatin (C-banding) and nucleolus organizer regions (NORs)]. Finally, although the methods to induce chromosome banding in most cases are only of modest technical difficulty, the correct identification of banded chromosomes (karyotyping) remains a tedious and very labor-intensive "art." Detailed discussion of the accepted criteria for identification of human chromosomes following banding analysis is beyond the scope of this chapter. Instead, the reader is referred to the recent International System for Cytogenetic Nomenclature (ISCN),4 a volume dedicated entirely to providing an international standard for identification and nomenclature of human chromosomes (similar standardized nomenclature committees have been formed and reports issued for other mammalian species).

1 J. Yunis, "Human Chromosome Methodology." Academic Press, New York, 1974.

2 H. Schwarzacher, "Methods in Human Cytogenetics." Springer-Verlag, New York, 1974.

3 S. Pathak and T. C. Hsu, Cytobios 43, 101 (1985).

4 "International System for Cytogenetic Nomenclature" (ISCN), p. 21. Karger, Basel, Switzerland, 1985.

Conventional Giemsa staining may be of considerable use in general descriptive studies of chromosomes from cell cultures. This simple staining procedure may be sufficient if the investigator is concerned only with simple documentation of chromosome number, or screening of metaPhosphate buffer: 0.06 A/KH2P04 (49 ml)/0.06 M Na2HP04 (51 ml)

1. Prepare a 4% Giemsa stain (in phosphate buffer pH to 6.8).

3. Rinse slides briefly with distilled water and air dry.

G-banding is one of the most useful and commonly employed banding procedures for identification of mammalian chromosomes. Advantages of this method over Q- or R-banding include the use of bright-field (rather than fluorescent) microscopy, and the semipermanent staining induced (allowing extended time to analyze or photograph a metaphase under the microscope). Several methods have been described for inducing G-banding, with most making use of either one or more proteolytic enzyme (e.g., trypsin), or mild heating of slides in a neutral buffer. In addition to identification of all normal chromosomes, G-banding is also very useful in identification of homogeneously staining regions (HSRs). However, G-banding is much less useful in identifying DMs because DMs stain lightly by G-banding and often can be overlooked. Three methods are described

Phosphate buffer: (see Standard Giemsa Staining) (pH 6.8) Giemsa stock stain: (Fisher, G-146 1 g, methanol 66 ml, and glycerin

5 N. Sun, E. Chu, and C. Chang, Mamm. Chromosome Newsl. 14, 26 (1973).

Staining procedure

1. Prepare a combined stain-enzyme solution as follows: (a) phosphate buffer 36.5 ml, (b) methanol (absolute) 12.5 ml, (c) Giemsa stain 0.9 ml, and (d) trypsin-EDTA (10X) 0.25 ml.

2. Preincubate slides for 10 min in a covered Coplin jar containing phosphate buffer prewarmed to 57°.

3. Remove slides from buffer and place horizontally on a staining rack. Gently cover the working surface of the slide with the stain-enzyme solution (~3 ml). (The viscosity of the stain-enzyme solution is sufficient to prevent drainage.) Incubate slides for 10-12 min at room temperature.

4. Rinse slides gently but thoroughly with distilled water and allow to air dry.

ASG Method6

Materials

Giemsa stock stain: [see Giemsa (G-)Banding]

Phosphate buffer: (see Standard Giemsa Staining) (pH 6.8)

Staining procedure

1. Preincubate slides in phosphate buffer at 56-60° for 8-12 min.

2. Rinse in distilled H20 and air dry.

3. Stain with Giemsa (4% in phosphate buffer) for 4 min.

4. Rinse in distilled H20 and air dry.

(If banding is not well differentiated, destain in 95% ethanol for 2-4 min, air dry, and repeat steps 3 and 4 using only 2-4 min staining.)

Wright Staining Method1

Materials

Phosphate buffer: (see Standard Giemsa Staining) (pH 6.8)

Wright stock stain: (0.25% in absolute methanol)

Staining procedure

1. Prepare Wright stain "working solution" as follows: (a) phosphate buffer, 3.0 ml, (b) Wright stock stain, 1.0 ml.

2. Make up fresh working solution for each slide.

3. Place slides on staining rack, flood slides with working solution, and stain for 1.5 to 2 min.

4. Rinse briefly with distilled H20.

5. If banding is not well differentiated, destain (as described below) and repeat steps 1 -4.

6 S. Schnedl, Chromosoma 34, 448 (1971).

7 J. Yunis, J. Sawyer, and D. Ball, Chromosoma 67, 293 (1978).

Destaining procedure: (a) preincubate slides in 95% ethanol for 1.5 min (with agitation) and air dry, (b) 95% ethanol + 1% HC1 for 30 sec with agitation and air dry, and (c) absolute methanol for 1.5 min and air dry.

Quinacrine (Q-)Banding*

Q-banding makes use of the DNA staining fluorochrome quinacrine mustard, or quinacrine dihydrochloride, to provide (like G-banding), unequivocal identification of all normal chromosomes (Fig. IB). Q-banding is in some regards more labor intensive than G-banding, due to the requirement of fluorescent (rather than bright-field microscopy) and the necessity to immediately photograph mitoses (due to quenching of the fluorochrome dye). Nevertheless, this technique has several advantages over G-banding, including (1) its ability to recognize more readily than G-banding polymorphisms of individual bands, (2) the overall "success rate" of Q-banding is superior to G-banding [especially in cases where metaphases are of marginal quality (e.g., tumor material)], and (3) most importantly, in cases where a minimum number of mitoses are available, Q-banding can be followed by a second banding technique (e.g., Q -* G; Q -* C) while the reverse order ordinarily cannot be followed. This ability to make use of sequential staining may be very important in identifying chromosomes which have undergone significant structural rearrangement.

Materials

Staining solution: Quinacrine mustard 2 mg/ml (in phosphate buffer)

Phosphate buffer: (see Standard Giemsa Staining) (pH to 5.5)

Ethanol

Mounting media: 10 g sucrose in 15 ml phosphate buffer (saturated solution)

Staining Procedure

1. Preincubate slides for 2 min each in 100% and then 50% ethanol.

2. Stain slides for 7-10 min.

3. Rinse slides for 2 min in phosphate buffer.

4. Mount slides using a #1 thickness coverslip (gently squeezing out excess mounting solution from under coverslip).

[Examination requires microscope with UV source and an excitation filter of 390-490 nm. Photography of fluorescence of Q-banded metaphases can be performed using Technical Pan film (Kodak) using manual exposures at 30 or 60 sec, and development in developer D-19 (Kodak) at 68° for 4 min.]

8 J. Caspersson, L. Zech, C. Johansson, and E. Modest, Chromosoma 30, 315 (1970).

Reverse (R-)Banding9

R-banding provides a "reverse" image of banding recognized by G- or Q-banding techniques. Specifically, the band pattern is opposite in staining intensity to G- or Q-bands with those chromosomal regions displaying very light Giemsa staining (or weak fluorescence) by G- or Q-banding, respectively, staining darkly by R-banding (and vice versa). This banding procedure has particular merit when a chromosomal alteration involves the terminal end of a chromosome (telomeric staining). However, the advantages of using R-banding over G- or Q-banding are minimal and this procedure is less often used than either G- or Q-banding techniques.

As with G-banding, numerous different procedures can be used to successfully induce R-bands in mammalian chromosomes. The following procedure is the one most often successful in our laboratory using established cell lines.

Materials

Staining solution: 0.04% Acridine Orange dye (in phosphate buffer pH 6.8)

Phosphate buffer: (see Standard Giemsa Staining) (pH to 6.8)

Mounting media: [see Quinacrine (Q-)Banding] (pH 6.8)

5-Bromodeoxyuridine (BrdUrd)

Stock solution: 100 /¿g/ml (in sterile distilled water) (wrap bottle in tin foil to protect from light)

Colcemid: 10 /¿g/ml in HBSS with phenol red (GIBCO)

Ethanol

Staining Procedure

Note: This method requires preincubation of cells with 5-BrdUrd in vitro prior to harvesting and staining of metaphases

1. Four to six hours prior to harvest of metaphases add BrdUrd to a final concentration of 10 ¿ug/ml (reincubate and protect from light).

2. One and one-half hours prior to harvest of metaphases, add colcemid to a final concentration of 0.05 jUg/ml (reincubate).

3. Harvest and prepare air-dried slides (see Procedures for Chromosome Procurement).

4. Place slides for 2 min each in 100% and then 50% ethanol.

5. Stain slides with Acridine Orange for 7-10 min.

6. Put slides through 2 rinses of 2 min each (with gentle agitation) in phosphate buffer.

7. Mount with #1 thickness coverslip, gently squeezing out excess working solution [see Quinacrine (Q-)Banding].

« M. Bobrow, P. Collacot, and K. Madan, Lancet 2, 1311 (1972).

[Examination requires excitation filter of 390-490 nm and addition of a barrier filter at 530 nm. Suggestions for photography of R-banded meta-phases include manual exposure time of 0.5, 1, or 2 sec using Tech Pan Film (Kodak) developed with D-19 developer (Kodak) at 68° for 4 min.]

Constitutive Heterochromatin (C-)Banding10

C-banding specifically stains areas of constitutive heterochromatin. While C-bands occur in all mammalian species, the amount and distribution may vary significantly between species. As is also true for NOR-band-ing, this pattern may be used effectively as a cytogenetic marker for species identification.3 In human cells, C-banding results in some pericentriomeric staining of all chromosomes, as well as staining of "secondary constrictions" found on chromosomes 1, 9, and 16 (Fig. 1C).

As with G-banding, the age of the slide should be taken into account in regards to incubation time. The minimum times presented represent the suggested time for fresh slides; maximum times are given for staining of slides up to 8 months old. This procedure is the most destructive to chromosome morphology of methods described in this report and should be used last in any attempts of sequential staining. Materials Barium hydroxide

Ba(OH)2 solution: Prepare a saturated solution of Ba(OH)2 and pour the resulting supernatant into a clean coplin jar prior to staining in order to reduce residue on slides. Phosphate buffer: (see Standard Giemsa Staining) (pH to 6.8) Giemsa stock stain: Gurr's R-66 Staining Procedure

1. Slides should be dipped twice in 95% ethanol, immediately dipped five to eight times in phosphate buffer, followed by placement in a saturated solution of Ba(OH)2 for 8 -11 min at room temperature.

2. Following exposure to Ba(OH)2, slides are dipped three times in 70% ethanol, three times in a second solution of phosphate buffer, and incubated in phosphate buffer at 60° for 4 hr.

3. Following incubation, slides are rinsed gently in tap water and stained for 5 min in 4% Giemsa (in phosphate buffer).

Silver Staining of Nucleolus Organizer Regions (NOR-)Bandingn

Staining of NORs recognizes transcriptionally active ribosomal cistrons in mammalian cells, with NOR's occurring in a species-specific pattern on

10 D. Miller, R. Tantranahi, V. Den, and O. J. Miller, Musculus Genet. 88, 67 (1976). " C. Goodpasture and S. Bloom, Chromosoma 53, 37 (1975).

mammalian chromosomes. In most species, NORs are found on several different chromosome pairs, with NORs in normal human cells restricted to the acrocentric (D and G group) chromosomes (Fig. ID). The silver staining method for NORs is particularly useful in examination of interspecies cross-culture contamination. The reader is referred to the manuscript of Pathak and Hsu3 for further discussion of interspecies patterns of NORs.

Materials

Silver nitrate solution: (50%) 1 g AgN03 in 2 ml distilled deionized water

0.45.//m filter syringe: (Millipore)

Giemsa stain: Gurr's R-66

Staining Procedure

It is essential that the same source of distilled deionized water be used for all steps in this procedure as this will greatly decrease the occurrence of nonspecific silver staining.

1. Prepare a 50% solution of silver nitrate and allow to stand at room temperature 15 min before use.

2. Incubate slides in distilled deionized water for 15 min at room temperature.

3. Prepare a moist chamber for silver incubation. (Use of square petri dish or plastic slide mailer containing moistened filter paper in the base is satisfactory.)

4. Remove slides from distilled water and allow to air dry.

5. Add 3-4 drops of the 50% silver nitrate solution onto each slide using a filter syringe (0.45 fim) and cover with a 22 X 40 mm coverglass.

6. Place slides into moist chamber, cover, and incubate at 56° for 8 -18 hr (the length of time may depend on the age of slides, with increased time for older preparations).

7. At the end of 18 hr, examine each slide for the presence of silver staining. You should observe a golden brown tint to the nuclear area and dark brown-black coloration of the nucleolar area. Dots of stained material should appear on the short arms of most of the acrocentric chromosomes (in human) at the conclusion of the incubation.

8. Following incubation, rinse slides with distilled deionized water and counterstain in 1% Giemsa stain for 7 sec.

(An alternate method to greatly decrease the time of incubation is to add three drops of 3% buffered formalin to the 50% AgN03 solution prior to staining. Slides may then be incubated for only 1 -4 hr at 65°.12

12 S. Pathak and F. Elder, Hum. Genet. 54, 171 (1980).

Procedures for Chromosome Procurement (Harvesting)

Harvesting of chromosomes for banding analysis has become routine, although some slight modifications must be made for monolayer versus suspension, or agar cultures. The general harvesting procedure will first be described, with modifications for monolayer suspension or agar cultures presented at the end of this section.

Materials

Colcemid: 10/ig/ml (GIBCO) Hypotonic solution: 0.075 MKC1 Fixative: methanol: glacial acetic acid (3:1)

Harvesting Procedure

1. Add Colcemid at a final concentration of 0.05 fig/m\ and reincubate 1.5 hr. (The optimal time of Colcemid exposure may vary for each cell line, although ordinarily longer colcemid exposures result in contracted chromosomes.)

2. After colcemid exposure, cells should be transferred into a 15 ml conical centrifuge tube and centrifuged at 600-800 rpm for 8-10 min.

3. Remove supernatant (keeping 0.2-0.5 ml) and resuspend cells with gentle aspiration using a Pasteur pipet (being careful not to aspirate cell suspension into the pipet as cell loss will occur onto the glass surface).

4. Add hypotonic 0.075 M KC1 (prewarmed to 37°) and resuspend cell-hypotonic solution. Incubate cells at 37° for 15-20 min (optimal time will depend on cells being studied).

5. Following hypotonic treatment, centrifuge cells at 600-800 rpm for 5 min. Remove supernatant (being careful not to disturb cell pellet) and resuspend cells in 5-10 ml of fresh cold fixative (3:1 methanol . glacial acetic acid) (suspend thoroughly but do not overpipet cells). Place cells at —20° for a minimum of 30 min.

6. Following Step 5, cells should be centrifuged and one additional change of fix should be made. After removing supernatant from the second fixative, cells should be resuspended in a small volume of fresh fixative (0.2-0.5 ml) for slide preparation.

7. Several procedures exist for air-dried slide preparation, the method described below is routinely utilized in our laboratory. Modifications to this procedure will often need to be made to account for changes in relative humidity and other factors related to individual laboratories.

a. Slides should be precleaned by placement for 30 min in absolute ethanol at —20°. After removing slides from the ethanol and draining off excess ethanol, slides should be allowed to air dry.

b. Three to five drops of cell suspension (from Step 6) should be dropped from a Pasteur pipet onto the cleaned microscope slide which is held at an approximate 45° angle (angling the slide will permit the suspension to run down the entire length of the slide, while increasing the distance from the pipet to the slide may assist in spreading of metaphases). A sharp burst of air can also be delivered to the slide to further enhance spreading.

c. Allow slides to air dry.

Cell Synchronization Procedure

Use of cell synchronization can be of great use in cytogenetic studies of human cell cultures.7 The advantages of cell synchronization are 2-fold: (1) the mitotic index of most cultures can be improved, and (2) harvesting of chromosomes can be optimized to capture cells closer to prophase than metaphase (resulting in greatly extended chromosomes). This later aspect in optimal preparations may increase the resolution of banding from around 450 total bands in metaphase chromosomes to the recognition of 850 or more bands in very uncondensed prometaphase chromosomes. Although decidedly more information is available in mitoses displaying 850 bands, the complexity of analysis of "high-resolution" banding also increases significantly. Accordingly, most of the work on high-resolution banding has been performed to date on human peripheral blood lymphocytes, where the kinetics of cell division have been thoroughly worked out. The nomenclature for high-resolution preparations of prophase and prometaphase human chromosomes is detailed within the ISCN.4

In our laboratory, the use of cell synchronization has been very helpful in obtaining metaphases from cultures with a very long generation time (e.g., early passage epithelial cell cultures). Although this procedure is not capable of providing an increase in the number or quality of metaphases from all cell cultures (particularly tumor cultures), it often is helpful in obtaining large numbers of mitoses of acceptable quality in cases where more standard techniques have failed.

Materials

Methotrexate stock solution: 10-5 (MW 454.46) (in H20)

Thymidine stock solution: 10-3 M (MW 242.2) (in H20)

Procedure

1. To block cells, add 10~7 M (final concentration) methotrexate to the culture medium and reincubate cell cultures for 16 -18 hr.

sterile distilled sterile distilled

2. To release cell blockage, remove the methotrexate containing medium, allow cells to stand for 5 min, and add fresh medium (without methotrexate), supplemented with 10-5 M (final concentration) thymidine, and reincubate for 5-7 hr.

3. Add Colcemid and harvest chromosomes as described in Procedures for Chromosome Procurement.

[The variable times of cell blockage and release must be established for each cell line. The ranges provided will work with a majority of established cell lines. Use of this method for primary tumor specimens is much more variable (due in part to the heterogeneous nature of primary tumor populations).]

Monolayer Cultures

Investigators should examine flasks before chromosome harvesting to insure adequate mitotic activity (evidenced by multiple rounded cells after mitotic arrest). In the event that mitoses are limited (even after prolonged colchicine exposure), cultures should be prepared using cell synchronization as described previously. Cells following mitotic arrest can be obtained mechanically by mitotic shake off or by using mild trypsinization.

Suspension Cultures

Harvesting procedure is identical to that described for monolayer cultures except there is no need to detach cells from plastic substrate.

Agar/Methylcellulose Cultures

Colony forming cells grown in agar/methylcellulose can be harvested for cytogenetic analysis with minor modifications of the procedure described for monolayer cultures.13,14 For examination of the entire plating layer (containing all colonies or clusters), 2 ml of media containing colchicine (10-7 M final concentration) is added, with reincubation for up to 16 hr (for human primary tumors) and, after removing agar layer containing the desired colonies, chromosome harvesting is accomplished as described previously. Alternately, single colonies can be plucked from the agar/ methylcellulose layer and harvested using micropipetting techniques.15

13 J. Trent, in "Cloning of Human Tumor Stem Cells," p. 345. Liss, New York, 1980.

151. O. Dube, C. J. Eaves, D. K. Kalousek, and A. C. Eaves, Cancer Genet. Cytogenet. 4,157 (1981).

Summary

Technological advances in the study of chromosomes from human and experimental cancers are occurring rapidly. Molecular cytogenetic techniques for in situ hybridization, as well as chromosome sorting and even karyotyping via flow cytometry (both described elsewhere within this volume), are important developing areas receiving considerable study. However, there currently remains a significant need for routine karyotyping of mammalian cells. It is hoped that the methods provided in this chapter will be of help in assisting somatic cell geneticists to identify chromosome changes in mammalian cell cultures.

Acknowledgments

Our thanks to Patricia Haight for excellent secretarial assistance. Dr. Trent is a scholar of the Leukemia Society of America. Sponsored in part by PHHS Grants CA-29476 and CA-17094.

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