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Uptake of isolated metaphase chromosomes by mammalian cells in vitro
Experimental Cell Research 61 (1970) 413-422
UPTAKE
OF ISOLATED
METAPHASE
MAMMALIAN
BY
CELLS IN VITRO
G. D. BURKHOLDER’ ‘Department
CHROMOSOMES
and B. B. MUKHERJEE’
of Medical Genetics, City of Hope Medical Center, Duarte, Calif, 91010, USA, and =Human Genetics Sector, Department of Genetics, McGill University, Montreal 110, Canada
SUMMARY In an autoradiographic investigation, it was found that isolated SH-thymidine-labelled metaphase chromosomes can penetrate into mammalian cells in vitro. The progressive changes in the cellular labelling patterns with time indicate that, in most cases, the ingested chromosome is degraded in the cytoplasm of the recipient cell and the chromosomal DNA is integrated into the host nuclear DNA. This integration of extracellular chromosomal DNA into the recipient cell DNA may involve DNA macromolecules rather than free nucleotides, and in most cases, the chromosomal DNA becomes randomly integrated among the chromosomes of the recipient cell. Rarely, whole extracellular chromosomes may become incorporated into a cell without being degraded, although this has not been established with certainty.
The development of successful methods for isolating mammalian metaphase chromosomes [2,3,6, 10, 121has focused considerable attention on the applications of isolated chromosomes to current problems in cell research. Chorazy et al. [4] originally reported that isolated metaphase chromosomes could penetrate into mammalian cells in vitro and obtained some evidence that the ingested chromosomal DNA might enter the recipient cell nucleus. More recently, Yosida & Sekiguchi [14] suggested that whole extracellular chromosomes became incorporated into the nuclei of recipient cells, and Ittensohn & Hutchison [7] have studied the cytological changes induced by chromosome uptake. Following the development of a modified technique for the isolation of mammalian metaphase chromosomes, the present research was undertaken to investigate, in detail, the events that occur during the uptake of
isolated metaphase chromosomes by cultured mammalian cells, and the fate of the ingested chromosomal DNA. MATERIALS
AND METHODS
Isolation of metaphase chromosomes For purposes of chromosome isolation, HeLa cells and L cells were maintained as monolayer cultures in Eagle minimal medium supplemented with glutamine (0.3 mg/ml) and 5 % each of calf serum and fetal bovine serum. Eight culture bottles, each with a surface area of 78 cm2. were routinely used for one isolation. In order to label the chromosomes, *H-thymidine (spec. act. 11.3 Ci/mM, Schwarz BioResearch) in a final concentration of 1 /Xi/ml was added to each culture for a neriod of 30 h urior to the collection of metaphase cells. When the-cells were approaching monolayer, the medium was removed from each culture and replaced with prewarmed calcium-deficient Eagle minimal medium supplemented with glutamine (0.3 mg/ml), 5 % each of calf serum and fetal bovine serum, and *H-thymidine (final concentration 1 &i/ml). Colcemid (CIBA) was then added to each culture in a final concentration of 0.5 pg/ml, in order to block the cells at metaphase. After incubation of the cells in this medium for 10 to 14 h, the cultures were gently shaken in a rocking fashion so that the Exptl Cell Res 61
4i4
G. D. Burkholder & B. B. Mukherjee
Fig. 1. Isolated metaphase chromosomes from (a) HeLa cells, x 700; and (b) L cells, x 800. Slide preparations
of isolated chromosomes were made by micropipetting a drop of a concentrated chromosome suspension onto a glass slide and air-drying. Dried preparations were subsequently fixed with 3 parts absolute ethanol: 1 part glacial acetic acid, and were stained with acetic orcein. medium flowed back and forth over the monolayer and metaphase cells were selectively detached from the glass surface. This “shake-off” procedure was initially described by Terasima & Tolmach [13], and Robbins & Marcus [ll]. Since cells become less firmly attached to the glass surface during metaphase, they may be relatively easily dislodged into the medium by gentle shaking. Utilizing this technique, cell suspensions were obtained in which 76 % of HeLa cells and 87 % of L cells were in metaphase. After the first metaphase collection, some cultures were supplied with fresh calcium-deficient medium and Colcemid, incubated for a further 10 to 14 h, and metaphase cells once more collected by the shakeoff technique. The metaphase cells were removed from the medium by centrifugation at 1 500 rpm for 5 min and were washed twice with Hanks balanced salt solution (BSS), centrifuging each time as before. Approx. 2 x lo6 - 4 x 10” cells were obtained from one series of shake-offs. The final pellet of washed cells was suspended in a hypotonic solution consisting of 1 part of Hanks BSS to 4 parts of distilled water to make a concentration of approx. 300 000 cells/ml. After a 30 min treatment at room temperature, the cells were removed from the hypotonic solution by centrifugation at 1 500 rpm for 5 min and the pellet was suspended in 10 c3 of an isolation medium consisting of 2 % citric acid, 0.1 M sucrose, 1O-s M CaCl,, lOA M MgC12, and 0.01 % Triton X-100, at 4°C. This isolation medium is a modification of the medium used by Salzman Exptl Cell Res 61
et al. [12]. In order to break open the cells, thereby releasing the metaphase chromosomes into solution, the suspension of cells in isolation medium was drawn into, and rapidly expelled from a 10 c3 syringe through an 18 gauge needle. This process was repeated until microscopic examination of a sample using phase contrast revealed that most of the cells had iysed. Examination of the cell lysate at this point revealed many single chromosomes, some small chromosome clumps, a few unbroken metaphases, interphase nuclei and some fine granular cytoplasmic debris. The lysate obtained from (2 x 10B)- (4 x 106) cells was diluted to annrox. 50 cs with a solution composed of 2 % ci& acid containing 0.1 M sucrose, 1O-s M CaCl,. 10m3 M M&l,. and 0.5 % Triton X-100, and was vigorously mixed with a Virtis 23 homogenizer. Separation of the interphase nuclei and clumped metaphases from free chromosomes was attained by filtration of the diluted homogenate through a fine sintered glass filter (Hysil brand) having a porosity of 20-30-pm. The solution was drawn through the filter by suction, using a vacuum pump. Nuclei and clumped metaphases become lodged in the pores of the filter while single chromosomes, which are considerably smaller, are able to pass through into the filtrate. The filtrate contained mostly single chromosomes and very few or no interphase nuclei (fig. la, bk
’ Purified chromosome preparations were morphologically stable in the isolation medium and could be stored for prolonged periods at 4°C.
Cellular
uptake of metaphase chromosomes
415
Fig. 2. Autoradiograms of L cells exposed to isolated 3H-thymidine-labelled L cell chromosomes: (a) compact clump of label attached to the edge of the cell membrane, x 1 600; (b) compact clump of label overlying the cytoplasm, x 1 600; (c) a semi-dispersed clump of grains over the cytoplasm, and dispersed grains over the nucleus, x 1 600; (d) residual grains over the cytoplasm, and dispersed grains over the nucleus, x I 600.
Preparation of isolated for cellular uptake
chromosomes
The chromosomes isolated from two or more shakeoffs were used for each uptake experiment. Although the chromosomes were not isolated under strict sterile conditions, it was found that the cell cultures to which chromosomes were added did not become contaminated during the short term cultivation periods reported here. The chromosomes were removed from the isolation medium by centrifugation at 2 400 rnm for 30 min. After discarding the sunernatant, the chromosomes were resuspended in 5 c3 of sterile 0.25 M sucrose and centrifuged as before. The resulting pellet was suspended in approx. 5 c3 of sterile Hanks BSS and centrifuged at 2 400 rpm for
30 min. The final chromosome pellet was suspended in 0.5 c3 of Hanks BSS and was added to the experimental culture. Cellular
uptake experiments
L cells, originally growing as monolayers, were seeded into calcium-deficient Eagle minimal medium supplemented with glutamine (0.3 mg/ml) and contaming 5 % each of fetal bovine serum and calf serum. A single 50Psuspension culture was established with an initial cell count of 146 000 cells/ml. After 20 h of incubation in a shaker bath, the cell concentration had reached 170 000 cells/ml. At this time. the culture was divided to form one 40 cs and one 10 c3 culture. The suspension of isolated L cell chromoExptl Cell Res 61
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G. D. Burkholder & B. B. Mukherjee
Table 1. Distribution
of labelling patterns in L cells exposed to L cell chromosomes at 37°C Clumps of label
Dispersed label
Hours of exposure to labelled chromosomes
Total no. labelled cells counted
Attached to edge of cell membrane
Over the cytoplasm
Over the nucleus
1 2 3 4 5 7 9 10 12
237 294 320 517 292 611 305 395 374
40.9a 34.1 26.8 28.1 35.2 23.4 23.3 23.7 18.2
50.2 58.5 61.4 62.3 55.5 64.5 65.2 63.7 63.1
8.9 6.8 9.1 5.2 6.3 5.0 3.9 3.4 6.1
Over the nucleus 0.0 0.0 2.7 4.4 3.0 7.1 7.6 9.2 12.6
a All values expressed as percentage of total number of labelled cells. somes, prepared as previously outlined, was added to the 40 c3 experimental culture (El), and as a control, 3H-thymidine was added to the 10 c3 culture (C) in a final concentration of approx. 0.066 ,&i/ml. Both cultures were mixed well and then 8 c3 of suspension was withdrawn from El and established separately (E2). El and C were maintained at 37°C in the shaker bath and E2 was kept at 1°C with periodic shaking. During a 12 h period, 2 c3 samples of El, C and E2 were withdrawn at 1, 2 and 3 h intervals respectively. The samples were centrifuged at 1 500 rpm for 5 min and the cell pellet was suspended in a few drops of distilled water, spread on a clean glass slide and gently dried at room temperature using an electric fan. After thorough drying, the slides were fixed in a mixture of 3 parts of absolute ethanol and 1 part of glacial acetic acid, and were later stained with acetic orcein. Ten hours after the addition of chromosomes to culture El, an 8 cs aliquot of cell suspension was removed. The cells were collected by centrifugation at 1 500 rpm for 5 min and were washed three times with prewarmed Hanks BSS in order to remove any unincorporated chromosomes remaining in the culture. Finally the cells wereresuspended in 10 c3of warm calcium-deficient Eagle minimal medium containing 5 % each of fetal bovine serum and calf serum, and Colcemid was added to the culture medium in a final concentration of 0.5 lug/ml. After 14 h of incubation in the shaker bath, the cells were treated with hypotonic solution (0.7 % sodium citrate), and fixative (3 parts absolute ethanol: 1 part glacial acetic acid). Chromosome preparations were made by flamedrying the cell suspension on glass slides and the chromosomes were stained with acetic orcein. Using a similar experimental design, isolated HeLa metaphase chromosomes were exposed to HeLa cells growing in suspension, or as monolayers in Leighton tubes.
Autoradiography
and scanning
All slides were coated with Kodak NTB3 liquid emulsion and were exposed in the dark for 4 weeks Exptl
Cell Res 61
before being developed. Developed slides were restained with 0.1 % Azur B. The slides from each experiment were scanned using a Zeiss photomicroscope. In order to avoid any bias when the cells with various labelling patterns were counted, the identifying code on each slide was covered, and remained concealed until all slides had been examined. All of the counts were made by one individual.
RESULTS After 1 h of exposure to labelled chromosomes, some of the autoradiographed cells were labelled with compact clumps of silver grains. These clumps could be seen attached to the outside edge of the cell membrane, overlying the cytoplasm or nucleus, or sometimes attached to the nuclear membrane (figs 2a, b; 4a). Most of the label associated with the cells at this time was in the form of compact masses of silver grains, however in a few cases, there seemed to be a “loosening” of the grains within the clumps, with evidence of some grain dispersion surrounding the central compact mass (fig. 4b). This apparent disintegration of the discrete clumps of silver grains was more frequently observed in later harvests. Microscopic examination of the autoradiographed cells serially harvested after chromosome addition, revealed a marked change in
Cellular uptake of metaphase chromosomes 417
Fig. 3. Autoradiograms of L cells exposed to isolated 3H-thymidine-labelled L cell chromosomes: (a) dispersed grains over the nucleus, and no cytoplasmic label, x 1 600; (b) randomly labelled metaphase figure obtained from cells exposed to labelled chromosomes for 10 h, washed, and recultured with Colcemid before harvesting, x 1 200; (c) labelled metaphase figure obtained from cells exposed to labelled chromosomes for IO h, washed, and recultured with Colcemid before harvesting. Note the clump of label over the figure (arrow), x 1 200; (d) control cell exposed to 3H-thymidine (0.066 ,&i/ml) for 6 h before harvesting, x 1 600.
the cellular distribution of label as the length of time that the cells were exposed to labelled chromosomes increased (table 1). Of the labelled cells observed in the sample harvested after 1 h exposure to extracellular chromosomes, 40.9 ~4 had a compact clump of grains attached to the edge of the cell membrane, and 50.2 y0 and 8.9 y0 had discrete clumps over the cytoplasm and nucleus, respectively. No cells were observed with completely randomly
dispersed grains. As the exposure time increased, the proportion of cells with label attached to the edge of the cell membrane gradually decreased, so that after 12 h the frequency of this labelling pattern had dropped to 18.2% (table 1). The frequency of cells with label over the cytoplasm gradually increased with time, while the number of cells with discrete clumps over the nuclei did not appear to change in any recognizable pattern. Exptl Cdl Res 61
418
G. D. Burkholder & B. B. Mukherjee
4d
Fig. 4. Autoradiograms of HeLa cells exposed to isolated 3H-thymidine-labelled HeLa cell chromosomes: (a) compact clump of label attached to the edge of the cell membrane, x 1200; (b) semi-dispersed grains over the cytoplasm, x 1 200; (c) compact clump of grains over the cytoplasm, and dispersed grains over the cytoplasm and nucleus, x 1 600; (d) dispersed grains over the nucleus, x 1 600; (e) randomly labelled metaphase figure obtained from cells exposed to labelled chromosomes for 9 h, washed, and recultured with Colcemid before harvesting, x 1 400; (f) compact clump of grains over a metaphase cell harvested directly from the experimental culture, x 1 400. ExptI Cell Res 61
Cellular uptake of metaphase chromosomes 419 Table 2. Distribution of labelling patterns in L cells exposed to L cell chromosomes at different temperatures Hours of exposure to labelled chromosomes 9 9 IO IO
Dispersed label
Clumps of label Temperature 1°C 37°C
Total no. labelled cells counted
Attached to edge of cell membrane
Over the cytoplasm
Over the nucleus
Over the nucleus
342 305 301 395
74.3a 23.3 73.7 23.7
21.6 65.2 22.6 63.7
4.1 3.9 3.1 3.4
0.0 7.6 0.0 9.2
a All values expressed as percentage of total number of labelled cells.
Dispersed nuclear label was first observed in the samples harvested 3 h after chromosomes were added to the cell cultures, when the frequency of this labelling pattern was 2.7 74 of all labelled cells. This random nuclear label was usually found in cells which were also labelled with loosened or semi-dispersed clumps of grains over the cytoplasm (figs 2c; 4~). Some cells with labelled nuclei had only a few, apparently residual, grains over the cytoplasm (fig. 2d), while others had diffuse label over the nucleus only and no cytoplasmic label (figs 3a; 4d). The proportion of cells labelled in this manner gradually increased with time, so that after 12 h of exposure to extracellular labelled chromosomes, the frequency of cells with diffuse nuclear label had increased to 12.6% (table 1). In the hourly harvests, made directly from the experimental (El) culture, most of the label was associated with interphase cells, but occasionally labelled metaphase cells were also seen. Rarely, a compact clump of grains was found overlying these metaphase cells (fig. 4f), but more frequently, dispersed label was located over the metaphase chromosomes. A number of labelled metaphase cells were observed in the culture that was incubated with labelled chromosomes for 10 h, washed to remove extracellular chromsomes, and re-
cultured with Colcemid for 14 h before harvesting. The label was randomly distributed among the chromosomes in most of the metaphase spreads, (figs 3b; 4e) however there were a few in which one chromosome appeared to be much more heavily labelled than the others (fig. 3~). If the cultures supplemented with labelled chromosomes were incubated at 1°C (experimental culture E2) instead of 37°C (El), a difference in the distribution of label was observed. Table 2 compares the distribution of label in samples withdrawn from El and E2 at corresponding times after chromosome feeding. For each pair of corresponding samples, the proportion of cells with discrete clumps of grains attached to the edge of the cell membrane was considerably higher (approx. 50 “/o) in the culture grown at 1°C than for that grown at 37°C and consequently there were fewer cells with cytoplasmic label in the 1°C culture than in the 37°C one. Virtually all of the labelled cells kept at 1°C exhibited compact clumps of silver grains, and unlike the cells maintained at 37°C rarely showed any loosened or semi-dispersed grain masses over the nucleus or cytoplasm, and never had any dispersed label overlying any part of the cell. The pattern of labelling observed in the control cultures (C) exposed to 3H-thymidine Exptl Cell Res 61
420 G. D. Burkholder & B. B. Mukherjee (0.066 ,&i/ml) was quite different from the patterns found in the experimental cultures (El and E2) incubated with extracellular labelled chromosomes. After 2 h of exposure to 3H-thymidine, a few of the cell nuclei were lightly labelled with randomly dispersed silver grains. As the exposure time increased however, both the proportion of nuclei labelled and the intensity of the label increased considerably. In all harvests, the silver grains were restricted to the cell nuclei, and were never seen over the cytoplasm (fig. 3d). The labelling patterns obtained in these experiments were not exclusive to L cells, but could also be obtained by administering labelled HeLa cell chromosomes to HeLa cells growing in suspension or as monolayers (fig. 4~3). DISCUSSION In this study, 3H-thymidine-labelled metaphase chromosomes were added to cells growing in vitro, and were the only source of radioactivity in the experimental cultures. Any label found associated with the cultured cells must therefore have originated from the labelled extracellular chromosomes. In a study of this nature, it is imperative to determine whether the isolated chromosomes actually penetrate into the cell or whether they merely adhere to the cell surface. Label attached to the outside edge of the cell membrane (figs 2~; 4a) obviously represents the attachment of chromosomes to the cell surface, but label overlying the cytoplasm (fig. 2b) or nucleus could represent either chromosomes that had been engulfed by the cell, or chromosomes attached to the cell surface. If the chromosomes adsorb to the cell membrane, but do not penetrate into the cells, there should not be any change in the cellular distribution of label as the length of time that the cells are exposed to labelled Exptl
Cell Res 61
chromosomes increases. The same proportion of cells with label attached to the edge of the cell membrane (or over the cytoplasm or nucleus) should be observed after 1 to 12 h of exposure. Examination of table 1 indicates that the distribution of label does not remain static however, but changes considerably as the exposure time increases. During a period of 12 h, the proportion of cells with compact label attached to the edge of the cell membrane drops from 40.9 % to 18.2 % while the proportion of cells with label over the cytoplasm increases from 50.2% to 65.2%. This change suggests that at least some of the adsorbed chromosomes are engulfed by the cells. Of interest is the observation that the decrease in the proportion of cells with label attached to the edge of the cell membrane seems to level off at the same time (7 h) that the increase in the proportion of cells with label over the cytoplasm reaches a plateau. These inverse changes in the cellular distribution of label would be expected if some of the adsorbed chromosomes are ingested by the cells. The course of chromosome uptake and the fate of the ingested chromosomal DNA was determined from the changes in the cellular labelling patterns with time. Following the adsorption of extracellular chromosomes to the cell surface, some of the adsorbed chromosomes pass from the extracellular environment into the cytoplasm of the cell. Phagocytosis is the most probable method by which chromosomes enter the recipient cell. Chorazy et al. [4] and Ittensohn & Hutchison [7] reported that the engulfed chromosomes were often located in cytoplasmic vacuoles. Many of the cells in the present study appeared to be highly vacuolated but because of the overlying label, it was difficult to tell whether the ingested chromosomes were actually located within vacuoles. If the process of chromosome uptake is
Cellular uptake of metaphase chromosomes 421 energy-dependent, fewer chromosomes would be expected to penetrate into cells maintained at low temperature since the over-all metabolism of the cell would be decreased. Table 2 shows that there is a dramatic difference in the distribution of label between cultures exposed to chromosomes at 37°C and 1°C for the same length of time. At the low temperature, a large proportion (approx. 74%) of the label is associated with the outside edge of the cell membrane, and smaller proportions with the cytoplasm or nucleus. At 37°C however, only a small proportion of the label is attached to the outside edge of the cell (approx. 23 “/). Most of the label is located over the cytoplasm. This difference in the distribution of label suggests that the incorporation process is energy-dependent. It appears that the chromosomes will readily adhere to the cell membrane at low temperature but do not easily penetrate into the cell. After a chromosome has entered into the cytoplasm of a cell, it is destroyed by the intracellular degradative enzymes. The progressive disintegration of the compact clumps of silver grains strongly suggested that the incorporated chromosomes were degraded within the cytoplasm. The first evidence of chromosome disintegration usually appeared 2 to 3 h after labelled chromosomes had been added to the cell cultures. At this time, some of the clumps of cellular label did not appear to be as compact as they were in earlier harvests. Sometimes condensed clumps of silver grains were surrounded by a few dispersed grains (fig. 4b). Later, in addition to these patterns, cells were found with both partially dispersed clumps of label over the cytoplasm, and randomly dispersed grains over the cytoplasm or nucleus, or both (figs 2c, d; 4~). From these patterns, it would appear that the ingested chromosome is degraded and the chromosomal products are released into the cytoplasm.
The disintegration of the discrete masses of label and concomitant appearance of diffuse label in the nucleus of the recipient cell suggested that the DNA of the disintegrated ingested chromosomes either became integrated into the nuclear DNA of the recipient cell, or entered the nucleus, but was not reutilized by the recipient cell. To distinguish between these two possibilities, cells were exposed to extracellular labelled chromosomes for several hours, washed to remove unincorporated chromosomes, and recultured in the presence of Colcemid to accumulate metaphase cells. Since the metaphase chromosomes of some of these cells were labelled (figs 3 b; 4e), it may be assumed that the chromosomal breakdown products had actually been integrated into the nuclear DNA of the recipient cell. The degradation of the engulfed chromosome by intracellular enzymes could result in either the complete disintegration of the chromosomal DNA to the level of free nucleotides, or incomplete breakdown to the level of polynucleotides. Thymidine, but not DNA, is soluble in ordinary acid fixatives such as the one used in the present study (3 : 1). After fixation with 3 : 1, the control cells, exposed to 3H-thymidine, were found to be labelled over the nucleus but not over the cytoplasm (fig. 3d). The fixative presumably removed the cytoplasmic acid-soluble 3H-thymidine pools from these cells, but did not affect the 3H-thymidine incorporated into the nuclear DNA. On the other hand, the experimental cells fixed in the same manner were often found to contain dispersed cytoplasmic label after several hours of exposure to labelled chromosomes (figs 2c, d, 4~). If complete disintegration of the DNA of the engulfed chromosome had occurred, and the free nucleotides had entered the cytoplasmic pool, the cytoplasmic label should have been removed by the fixative. Since it was not, this Exptl Cell Res 61
422
G. D. Burkholder & B. B. Mukherjee
cytoplasmic label must represent segments of DNA rather than free nucleotides. The present experiments do not definitely determine whether the nuclear label results from the integration of individual nucleotides, or of polynucleotides, into the recipient cell DNA, however in view of the fact that extracellular DNA can penetrate into cells, and become integrated into the recipient cell genome in a macromolecular form [I, 5, 8, 91, there appears to be no reason why DNA molecules released from engulfed chromosomes could not be incorporated into the nuclear DNA of the recipient cell. Assuming that polynucleotides of chromosomal DNA can be integrated into the recipient cell DNA, it appears feasible that new or different genes could be introduced into the genome of the recipient cell. If the newly integrated genes were functional, new cellular characteristics might be acquired, and genetic transformation would occur. Most of the labelling patterns indicated that the engulfed chromosome was degraded in the cytoplasm and the breakdown products were integrated into the nuclear DNA. In many of the labelled metaphase cells accumulated with Colcemid after exposure to labelled chromosomes, the silver grains, and hence the degraded chromosome products, were randomly distributed among most of the metaphase chromosomes (figs 3 b; 4e). There were a few cells however, where one chromosome was more heavily labelled than the rest (fig. 3~). There may be two explanations for this. Whole extracellular chromosomes may, on occasion, become incorporated into a cell without being degraded. The most likely time for this to occur would be when the cell was undergoing mitosis. Since the cell’s own metaphase chromosomes are not destroyed during the division phase, when they exist free in the cytoplasm, an incorporated chromosome may also escape destrucExptl
Cell Res 61
tion and might be enclosed within one of the new nuclei formed at telophase. Unfortunately, since the cell lines used in this study were heteroploid, and the chromosome number was quite variable from cell to cell, karyotype analysis would not detect the presence of a ‘new’ cellular chromosome. Alternatively however, an extracellular chromosome might be engulfed and degraded in the cytoplasm in the usual manner, but instead of the breakdown products becoming randomly integrated into the cellular chromosomes, this DNA might be specifically incorporated by the corresponding chromosome within the recipient cell. This work forms part of a doctoral dissertation submitted by G. D. Burkholder to the Faculty of Graduate Studies and Research, McGill University. The excellent technical assistance of Mr David M. Sigman, and competent secretarial help of Mrs. L. Ramaglia are gratefully acknowledged. This investigation was supported by grant MT2169 from the Medical Research Council of Canada, and a postgraduate scholarship from the National Research Council of Canada.
REFERENCES Ayad, S R & Fox, M, Nature 220 (1968) 35. Cantor, K P & Hearst, J E, Proc natl acad sci US 55 (1966) 642. Chorazy, M, Bendich, A, Borenfreund, E & Hutchison, D J, J cell biol 19 (1963) 59. Chorazy, M, Bendich, A, Borenfreund, E, Ittensohn, 0 L & Hutchison, D J, J cell biol 19 (1963) 71. 5. Gartler, S M, Biochem biophys res comm 3 (1960) 127. 6. Huberman, J A & Attardi, G, J cell biol 31 (1966) 95. I. Ittensohn, 0 L & Hutchison, D J, Exptl cell res 55 (1969) 149. 8. Kay, E R M, Trans NY acad sci 28 (1966) 726. 9. Ledoux, L, Progr nucl acid res 4 (1965) 231. 10. Maio, J J & Schildkraut, C L, Methods in cell physiology (ed D M Prescott) vol. 2, p. 113. Academic Press, New York (1966). 11. Robbins. E & Marcus, P I. Science 144 (I 964) 1152. 12. Salzman, N P, Moore, D E & Mendelsohn, J, Proc natl acad sci US 56 (1966) 1449. 13. Terasima, T & Tolmach,‘L J,’ Exptl cell res 30 (1963) 344. 14. Yosida, T H & Sekiguchi, T, Mol and gen genet 103 (1968) 253. Received February 23, 1970
UPTAKE
OF ISOLATED
METAPHASE
MAMMALIAN
BY
CELLS IN VITRO
G. D. BURKHOLDER’ ‘Department
CHROMOSOMES
and B. B. MUKHERJEE’
of Medical Genetics, City of Hope Medical Center, Duarte, Calif, 91010, USA, and =Human Genetics Sector, Department of Genetics, McGill University, Montreal 110, Canada
SUMMARY In an autoradiographic investigation, it was found that isolated SH-thymidine-labelled metaphase chromosomes can penetrate into mammalian cells in vitro. The progressive changes in the cellular labelling patterns with time indicate that, in most cases, the ingested chromosome is degraded in the cytoplasm of the recipient cell and the chromosomal DNA is integrated into the host nuclear DNA. This integration of extracellular chromosomal DNA into the recipient cell DNA may involve DNA macromolecules rather than free nucleotides, and in most cases, the chromosomal DNA becomes randomly integrated among the chromosomes of the recipient cell. Rarely, whole extracellular chromosomes may become incorporated into a cell without being degraded, although this has not been established with certainty.
The development of successful methods for isolating mammalian metaphase chromosomes [2,3,6, 10, 121has focused considerable attention on the applications of isolated chromosomes to current problems in cell research. Chorazy et al. [4] originally reported that isolated metaphase chromosomes could penetrate into mammalian cells in vitro and obtained some evidence that the ingested chromosomal DNA might enter the recipient cell nucleus. More recently, Yosida & Sekiguchi [14] suggested that whole extracellular chromosomes became incorporated into the nuclei of recipient cells, and Ittensohn & Hutchison [7] have studied the cytological changes induced by chromosome uptake. Following the development of a modified technique for the isolation of mammalian metaphase chromosomes, the present research was undertaken to investigate, in detail, the events that occur during the uptake of
isolated metaphase chromosomes by cultured mammalian cells, and the fate of the ingested chromosomal DNA. MATERIALS
AND METHODS
Isolation of metaphase chromosomes For purposes of chromosome isolation, HeLa cells and L cells were maintained as monolayer cultures in Eagle minimal medium supplemented with glutamine (0.3 mg/ml) and 5 % each of calf serum and fetal bovine serum. Eight culture bottles, each with a surface area of 78 cm2. were routinely used for one isolation. In order to label the chromosomes, *H-thymidine (spec. act. 11.3 Ci/mM, Schwarz BioResearch) in a final concentration of 1 /Xi/ml was added to each culture for a neriod of 30 h urior to the collection of metaphase cells. When the-cells were approaching monolayer, the medium was removed from each culture and replaced with prewarmed calcium-deficient Eagle minimal medium supplemented with glutamine (0.3 mg/ml), 5 % each of calf serum and fetal bovine serum, and *H-thymidine (final concentration 1 &i/ml). Colcemid (CIBA) was then added to each culture in a final concentration of 0.5 pg/ml, in order to block the cells at metaphase. After incubation of the cells in this medium for 10 to 14 h, the cultures were gently shaken in a rocking fashion so that the Exptl Cell Res 61
4i4
G. D. Burkholder & B. B. Mukherjee
Fig. 1. Isolated metaphase chromosomes from (a) HeLa cells, x 700; and (b) L cells, x 800. Slide preparations
of isolated chromosomes were made by micropipetting a drop of a concentrated chromosome suspension onto a glass slide and air-drying. Dried preparations were subsequently fixed with 3 parts absolute ethanol: 1 part glacial acetic acid, and were stained with acetic orcein. medium flowed back and forth over the monolayer and metaphase cells were selectively detached from the glass surface. This “shake-off” procedure was initially described by Terasima & Tolmach [13], and Robbins & Marcus [ll]. Since cells become less firmly attached to the glass surface during metaphase, they may be relatively easily dislodged into the medium by gentle shaking. Utilizing this technique, cell suspensions were obtained in which 76 % of HeLa cells and 87 % of L cells were in metaphase. After the first metaphase collection, some cultures were supplied with fresh calcium-deficient medium and Colcemid, incubated for a further 10 to 14 h, and metaphase cells once more collected by the shakeoff technique. The metaphase cells were removed from the medium by centrifugation at 1 500 rpm for 5 min and were washed twice with Hanks balanced salt solution (BSS), centrifuging each time as before. Approx. 2 x lo6 - 4 x 10” cells were obtained from one series of shake-offs. The final pellet of washed cells was suspended in a hypotonic solution consisting of 1 part of Hanks BSS to 4 parts of distilled water to make a concentration of approx. 300 000 cells/ml. After a 30 min treatment at room temperature, the cells were removed from the hypotonic solution by centrifugation at 1 500 rpm for 5 min and the pellet was suspended in 10 c3 of an isolation medium consisting of 2 % citric acid, 0.1 M sucrose, 1O-s M CaCl,, lOA M MgC12, and 0.01 % Triton X-100, at 4°C. This isolation medium is a modification of the medium used by Salzman Exptl Cell Res 61
et al. [12]. In order to break open the cells, thereby releasing the metaphase chromosomes into solution, the suspension of cells in isolation medium was drawn into, and rapidly expelled from a 10 c3 syringe through an 18 gauge needle. This process was repeated until microscopic examination of a sample using phase contrast revealed that most of the cells had iysed. Examination of the cell lysate at this point revealed many single chromosomes, some small chromosome clumps, a few unbroken metaphases, interphase nuclei and some fine granular cytoplasmic debris. The lysate obtained from (2 x 10B)- (4 x 106) cells was diluted to annrox. 50 cs with a solution composed of 2 % ci& acid containing 0.1 M sucrose, 1O-s M CaCl,. 10m3 M M&l,. and 0.5 % Triton X-100, and was vigorously mixed with a Virtis 23 homogenizer. Separation of the interphase nuclei and clumped metaphases from free chromosomes was attained by filtration of the diluted homogenate through a fine sintered glass filter (Hysil brand) having a porosity of 20-30-pm. The solution was drawn through the filter by suction, using a vacuum pump. Nuclei and clumped metaphases become lodged in the pores of the filter while single chromosomes, which are considerably smaller, are able to pass through into the filtrate. The filtrate contained mostly single chromosomes and very few or no interphase nuclei (fig. la, bk
’ Purified chromosome preparations were morphologically stable in the isolation medium and could be stored for prolonged periods at 4°C.
Cellular
uptake of metaphase chromosomes
415
Fig. 2. Autoradiograms of L cells exposed to isolated 3H-thymidine-labelled L cell chromosomes: (a) compact clump of label attached to the edge of the cell membrane, x 1 600; (b) compact clump of label overlying the cytoplasm, x 1 600; (c) a semi-dispersed clump of grains over the cytoplasm, and dispersed grains over the nucleus, x 1 600; (d) residual grains over the cytoplasm, and dispersed grains over the nucleus, x I 600.
Preparation of isolated for cellular uptake
chromosomes
The chromosomes isolated from two or more shakeoffs were used for each uptake experiment. Although the chromosomes were not isolated under strict sterile conditions, it was found that the cell cultures to which chromosomes were added did not become contaminated during the short term cultivation periods reported here. The chromosomes were removed from the isolation medium by centrifugation at 2 400 rnm for 30 min. After discarding the sunernatant, the chromosomes were resuspended in 5 c3 of sterile 0.25 M sucrose and centrifuged as before. The resulting pellet was suspended in approx. 5 c3 of sterile Hanks BSS and centrifuged at 2 400 rpm for
30 min. The final chromosome pellet was suspended in 0.5 c3 of Hanks BSS and was added to the experimental culture. Cellular
uptake experiments
L cells, originally growing as monolayers, were seeded into calcium-deficient Eagle minimal medium supplemented with glutamine (0.3 mg/ml) and contaming 5 % each of fetal bovine serum and calf serum. A single 50Psuspension culture was established with an initial cell count of 146 000 cells/ml. After 20 h of incubation in a shaker bath, the cell concentration had reached 170 000 cells/ml. At this time. the culture was divided to form one 40 cs and one 10 c3 culture. The suspension of isolated L cell chromoExptl Cell Res 61
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Table 1. Distribution
of labelling patterns in L cells exposed to L cell chromosomes at 37°C Clumps of label
Dispersed label
Hours of exposure to labelled chromosomes
Total no. labelled cells counted
Attached to edge of cell membrane
Over the cytoplasm
Over the nucleus
1 2 3 4 5 7 9 10 12
237 294 320 517 292 611 305 395 374
40.9a 34.1 26.8 28.1 35.2 23.4 23.3 23.7 18.2
50.2 58.5 61.4 62.3 55.5 64.5 65.2 63.7 63.1
8.9 6.8 9.1 5.2 6.3 5.0 3.9 3.4 6.1
Over the nucleus 0.0 0.0 2.7 4.4 3.0 7.1 7.6 9.2 12.6
a All values expressed as percentage of total number of labelled cells. somes, prepared as previously outlined, was added to the 40 c3 experimental culture (El), and as a control, 3H-thymidine was added to the 10 c3 culture (C) in a final concentration of approx. 0.066 ,&i/ml. Both cultures were mixed well and then 8 c3 of suspension was withdrawn from El and established separately (E2). El and C were maintained at 37°C in the shaker bath and E2 was kept at 1°C with periodic shaking. During a 12 h period, 2 c3 samples of El, C and E2 were withdrawn at 1, 2 and 3 h intervals respectively. The samples were centrifuged at 1 500 rpm for 5 min and the cell pellet was suspended in a few drops of distilled water, spread on a clean glass slide and gently dried at room temperature using an electric fan. After thorough drying, the slides were fixed in a mixture of 3 parts of absolute ethanol and 1 part of glacial acetic acid, and were later stained with acetic orcein. Ten hours after the addition of chromosomes to culture El, an 8 cs aliquot of cell suspension was removed. The cells were collected by centrifugation at 1 500 rpm for 5 min and were washed three times with prewarmed Hanks BSS in order to remove any unincorporated chromosomes remaining in the culture. Finally the cells wereresuspended in 10 c3of warm calcium-deficient Eagle minimal medium containing 5 % each of fetal bovine serum and calf serum, and Colcemid was added to the culture medium in a final concentration of 0.5 lug/ml. After 14 h of incubation in the shaker bath, the cells were treated with hypotonic solution (0.7 % sodium citrate), and fixative (3 parts absolute ethanol: 1 part glacial acetic acid). Chromosome preparations were made by flamedrying the cell suspension on glass slides and the chromosomes were stained with acetic orcein. Using a similar experimental design, isolated HeLa metaphase chromosomes were exposed to HeLa cells growing in suspension, or as monolayers in Leighton tubes.
Autoradiography
and scanning
All slides were coated with Kodak NTB3 liquid emulsion and were exposed in the dark for 4 weeks Exptl
Cell Res 61
before being developed. Developed slides were restained with 0.1 % Azur B. The slides from each experiment were scanned using a Zeiss photomicroscope. In order to avoid any bias when the cells with various labelling patterns were counted, the identifying code on each slide was covered, and remained concealed until all slides had been examined. All of the counts were made by one individual.
RESULTS After 1 h of exposure to labelled chromosomes, some of the autoradiographed cells were labelled with compact clumps of silver grains. These clumps could be seen attached to the outside edge of the cell membrane, overlying the cytoplasm or nucleus, or sometimes attached to the nuclear membrane (figs 2a, b; 4a). Most of the label associated with the cells at this time was in the form of compact masses of silver grains, however in a few cases, there seemed to be a “loosening” of the grains within the clumps, with evidence of some grain dispersion surrounding the central compact mass (fig. 4b). This apparent disintegration of the discrete clumps of silver grains was more frequently observed in later harvests. Microscopic examination of the autoradiographed cells serially harvested after chromosome addition, revealed a marked change in
Cellular uptake of metaphase chromosomes 417
Fig. 3. Autoradiograms of L cells exposed to isolated 3H-thymidine-labelled L cell chromosomes: (a) dispersed grains over the nucleus, and no cytoplasmic label, x 1 600; (b) randomly labelled metaphase figure obtained from cells exposed to labelled chromosomes for 10 h, washed, and recultured with Colcemid before harvesting, x 1 200; (c) labelled metaphase figure obtained from cells exposed to labelled chromosomes for IO h, washed, and recultured with Colcemid before harvesting. Note the clump of label over the figure (arrow), x 1 200; (d) control cell exposed to 3H-thymidine (0.066 ,&i/ml) for 6 h before harvesting, x 1 600.
the cellular distribution of label as the length of time that the cells were exposed to labelled chromosomes increased (table 1). Of the labelled cells observed in the sample harvested after 1 h exposure to extracellular chromosomes, 40.9 ~4 had a compact clump of grains attached to the edge of the cell membrane, and 50.2 y0 and 8.9 y0 had discrete clumps over the cytoplasm and nucleus, respectively. No cells were observed with completely randomly
dispersed grains. As the exposure time increased, the proportion of cells with label attached to the edge of the cell membrane gradually decreased, so that after 12 h the frequency of this labelling pattern had dropped to 18.2% (table 1). The frequency of cells with label over the cytoplasm gradually increased with time, while the number of cells with discrete clumps over the nuclei did not appear to change in any recognizable pattern. Exptl Cdl Res 61
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4d
Fig. 4. Autoradiograms of HeLa cells exposed to isolated 3H-thymidine-labelled HeLa cell chromosomes: (a) compact clump of label attached to the edge of the cell membrane, x 1200; (b) semi-dispersed grains over the cytoplasm, x 1 200; (c) compact clump of grains over the cytoplasm, and dispersed grains over the cytoplasm and nucleus, x 1 600; (d) dispersed grains over the nucleus, x 1 600; (e) randomly labelled metaphase figure obtained from cells exposed to labelled chromosomes for 9 h, washed, and recultured with Colcemid before harvesting, x 1 400; (f) compact clump of grains over a metaphase cell harvested directly from the experimental culture, x 1 400. ExptI Cell Res 61
Cellular uptake of metaphase chromosomes 419 Table 2. Distribution of labelling patterns in L cells exposed to L cell chromosomes at different temperatures Hours of exposure to labelled chromosomes 9 9 IO IO
Dispersed label
Clumps of label Temperature 1°C 37°C
Total no. labelled cells counted
Attached to edge of cell membrane
Over the cytoplasm
Over the nucleus
Over the nucleus
342 305 301 395
74.3a 23.3 73.7 23.7
21.6 65.2 22.6 63.7
4.1 3.9 3.1 3.4
0.0 7.6 0.0 9.2
a All values expressed as percentage of total number of labelled cells.
Dispersed nuclear label was first observed in the samples harvested 3 h after chromosomes were added to the cell cultures, when the frequency of this labelling pattern was 2.7 74 of all labelled cells. This random nuclear label was usually found in cells which were also labelled with loosened or semi-dispersed clumps of grains over the cytoplasm (figs 2c; 4~). Some cells with labelled nuclei had only a few, apparently residual, grains over the cytoplasm (fig. 2d), while others had diffuse label over the nucleus only and no cytoplasmic label (figs 3a; 4d). The proportion of cells labelled in this manner gradually increased with time, so that after 12 h of exposure to extracellular labelled chromosomes, the frequency of cells with diffuse nuclear label had increased to 12.6% (table 1). In the hourly harvests, made directly from the experimental (El) culture, most of the label was associated with interphase cells, but occasionally labelled metaphase cells were also seen. Rarely, a compact clump of grains was found overlying these metaphase cells (fig. 4f), but more frequently, dispersed label was located over the metaphase chromosomes. A number of labelled metaphase cells were observed in the culture that was incubated with labelled chromosomes for 10 h, washed to remove extracellular chromsomes, and re-
cultured with Colcemid for 14 h before harvesting. The label was randomly distributed among the chromosomes in most of the metaphase spreads, (figs 3b; 4e) however there were a few in which one chromosome appeared to be much more heavily labelled than the others (fig. 3~). If the cultures supplemented with labelled chromosomes were incubated at 1°C (experimental culture E2) instead of 37°C (El), a difference in the distribution of label was observed. Table 2 compares the distribution of label in samples withdrawn from El and E2 at corresponding times after chromosome feeding. For each pair of corresponding samples, the proportion of cells with discrete clumps of grains attached to the edge of the cell membrane was considerably higher (approx. 50 “/o) in the culture grown at 1°C than for that grown at 37°C and consequently there were fewer cells with cytoplasmic label in the 1°C culture than in the 37°C one. Virtually all of the labelled cells kept at 1°C exhibited compact clumps of silver grains, and unlike the cells maintained at 37°C rarely showed any loosened or semi-dispersed grain masses over the nucleus or cytoplasm, and never had any dispersed label overlying any part of the cell. The pattern of labelling observed in the control cultures (C) exposed to 3H-thymidine Exptl Cell Res 61
420 G. D. Burkholder & B. B. Mukherjee (0.066 ,&i/ml) was quite different from the patterns found in the experimental cultures (El and E2) incubated with extracellular labelled chromosomes. After 2 h of exposure to 3H-thymidine, a few of the cell nuclei were lightly labelled with randomly dispersed silver grains. As the exposure time increased however, both the proportion of nuclei labelled and the intensity of the label increased considerably. In all harvests, the silver grains were restricted to the cell nuclei, and were never seen over the cytoplasm (fig. 3d). The labelling patterns obtained in these experiments were not exclusive to L cells, but could also be obtained by administering labelled HeLa cell chromosomes to HeLa cells growing in suspension or as monolayers (fig. 4~3). DISCUSSION In this study, 3H-thymidine-labelled metaphase chromosomes were added to cells growing in vitro, and were the only source of radioactivity in the experimental cultures. Any label found associated with the cultured cells must therefore have originated from the labelled extracellular chromosomes. In a study of this nature, it is imperative to determine whether the isolated chromosomes actually penetrate into the cell or whether they merely adhere to the cell surface. Label attached to the outside edge of the cell membrane (figs 2~; 4a) obviously represents the attachment of chromosomes to the cell surface, but label overlying the cytoplasm (fig. 2b) or nucleus could represent either chromosomes that had been engulfed by the cell, or chromosomes attached to the cell surface. If the chromosomes adsorb to the cell membrane, but do not penetrate into the cells, there should not be any change in the cellular distribution of label as the length of time that the cells are exposed to labelled Exptl
Cell Res 61
chromosomes increases. The same proportion of cells with label attached to the edge of the cell membrane (or over the cytoplasm or nucleus) should be observed after 1 to 12 h of exposure. Examination of table 1 indicates that the distribution of label does not remain static however, but changes considerably as the exposure time increases. During a period of 12 h, the proportion of cells with compact label attached to the edge of the cell membrane drops from 40.9 % to 18.2 % while the proportion of cells with label over the cytoplasm increases from 50.2% to 65.2%. This change suggests that at least some of the adsorbed chromosomes are engulfed by the cells. Of interest is the observation that the decrease in the proportion of cells with label attached to the edge of the cell membrane seems to level off at the same time (7 h) that the increase in the proportion of cells with label over the cytoplasm reaches a plateau. These inverse changes in the cellular distribution of label would be expected if some of the adsorbed chromosomes are ingested by the cells. The course of chromosome uptake and the fate of the ingested chromosomal DNA was determined from the changes in the cellular labelling patterns with time. Following the adsorption of extracellular chromosomes to the cell surface, some of the adsorbed chromosomes pass from the extracellular environment into the cytoplasm of the cell. Phagocytosis is the most probable method by which chromosomes enter the recipient cell. Chorazy et al. [4] and Ittensohn & Hutchison [7] reported that the engulfed chromosomes were often located in cytoplasmic vacuoles. Many of the cells in the present study appeared to be highly vacuolated but because of the overlying label, it was difficult to tell whether the ingested chromosomes were actually located within vacuoles. If the process of chromosome uptake is
Cellular uptake of metaphase chromosomes 421 energy-dependent, fewer chromosomes would be expected to penetrate into cells maintained at low temperature since the over-all metabolism of the cell would be decreased. Table 2 shows that there is a dramatic difference in the distribution of label between cultures exposed to chromosomes at 37°C and 1°C for the same length of time. At the low temperature, a large proportion (approx. 74%) of the label is associated with the outside edge of the cell membrane, and smaller proportions with the cytoplasm or nucleus. At 37°C however, only a small proportion of the label is attached to the outside edge of the cell (approx. 23 “/). Most of the label is located over the cytoplasm. This difference in the distribution of label suggests that the incorporation process is energy-dependent. It appears that the chromosomes will readily adhere to the cell membrane at low temperature but do not easily penetrate into the cell. After a chromosome has entered into the cytoplasm of a cell, it is destroyed by the intracellular degradative enzymes. The progressive disintegration of the compact clumps of silver grains strongly suggested that the incorporated chromosomes were degraded within the cytoplasm. The first evidence of chromosome disintegration usually appeared 2 to 3 h after labelled chromosomes had been added to the cell cultures. At this time, some of the clumps of cellular label did not appear to be as compact as they were in earlier harvests. Sometimes condensed clumps of silver grains were surrounded by a few dispersed grains (fig. 4b). Later, in addition to these patterns, cells were found with both partially dispersed clumps of label over the cytoplasm, and randomly dispersed grains over the cytoplasm or nucleus, or both (figs 2c, d; 4~). From these patterns, it would appear that the ingested chromosome is degraded and the chromosomal products are released into the cytoplasm.
The disintegration of the discrete masses of label and concomitant appearance of diffuse label in the nucleus of the recipient cell suggested that the DNA of the disintegrated ingested chromosomes either became integrated into the nuclear DNA of the recipient cell, or entered the nucleus, but was not reutilized by the recipient cell. To distinguish between these two possibilities, cells were exposed to extracellular labelled chromosomes for several hours, washed to remove unincorporated chromosomes, and recultured in the presence of Colcemid to accumulate metaphase cells. Since the metaphase chromosomes of some of these cells were labelled (figs 3 b; 4e), it may be assumed that the chromosomal breakdown products had actually been integrated into the nuclear DNA of the recipient cell. The degradation of the engulfed chromosome by intracellular enzymes could result in either the complete disintegration of the chromosomal DNA to the level of free nucleotides, or incomplete breakdown to the level of polynucleotides. Thymidine, but not DNA, is soluble in ordinary acid fixatives such as the one used in the present study (3 : 1). After fixation with 3 : 1, the control cells, exposed to 3H-thymidine, were found to be labelled over the nucleus but not over the cytoplasm (fig. 3d). The fixative presumably removed the cytoplasmic acid-soluble 3H-thymidine pools from these cells, but did not affect the 3H-thymidine incorporated into the nuclear DNA. On the other hand, the experimental cells fixed in the same manner were often found to contain dispersed cytoplasmic label after several hours of exposure to labelled chromosomes (figs 2c, d, 4~). If complete disintegration of the DNA of the engulfed chromosome had occurred, and the free nucleotides had entered the cytoplasmic pool, the cytoplasmic label should have been removed by the fixative. Since it was not, this Exptl Cell Res 61
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G. D. Burkholder & B. B. Mukherjee
cytoplasmic label must represent segments of DNA rather than free nucleotides. The present experiments do not definitely determine whether the nuclear label results from the integration of individual nucleotides, or of polynucleotides, into the recipient cell DNA, however in view of the fact that extracellular DNA can penetrate into cells, and become integrated into the recipient cell genome in a macromolecular form [I, 5, 8, 91, there appears to be no reason why DNA molecules released from engulfed chromosomes could not be incorporated into the nuclear DNA of the recipient cell. Assuming that polynucleotides of chromosomal DNA can be integrated into the recipient cell DNA, it appears feasible that new or different genes could be introduced into the genome of the recipient cell. If the newly integrated genes were functional, new cellular characteristics might be acquired, and genetic transformation would occur. Most of the labelling patterns indicated that the engulfed chromosome was degraded in the cytoplasm and the breakdown products were integrated into the nuclear DNA. In many of the labelled metaphase cells accumulated with Colcemid after exposure to labelled chromosomes, the silver grains, and hence the degraded chromosome products, were randomly distributed among most of the metaphase chromosomes (figs 3 b; 4e). There were a few cells however, where one chromosome was more heavily labelled than the rest (fig. 3~). There may be two explanations for this. Whole extracellular chromosomes may, on occasion, become incorporated into a cell without being degraded. The most likely time for this to occur would be when the cell was undergoing mitosis. Since the cell’s own metaphase chromosomes are not destroyed during the division phase, when they exist free in the cytoplasm, an incorporated chromosome may also escape destrucExptl
Cell Res 61
tion and might be enclosed within one of the new nuclei formed at telophase. Unfortunately, since the cell lines used in this study were heteroploid, and the chromosome number was quite variable from cell to cell, karyotype analysis would not detect the presence of a ‘new’ cellular chromosome. Alternatively however, an extracellular chromosome might be engulfed and degraded in the cytoplasm in the usual manner, but instead of the breakdown products becoming randomly integrated into the cellular chromosomes, this DNA might be specifically incorporated by the corresponding chromosome within the recipient cell. This work forms part of a doctoral dissertation submitted by G. D. Burkholder to the Faculty of Graduate Studies and Research, McGill University. The excellent technical assistance of Mr David M. Sigman, and competent secretarial help of Mrs. L. Ramaglia are gratefully acknowledged. This investigation was supported by grant MT2169 from the Medical Research Council of Canada, and a postgraduate scholarship from the National Research Council of Canada.
REFERENCES Ayad, S R & Fox, M, Nature 220 (1968) 35. Cantor, K P & Hearst, J E, Proc natl acad sci US 55 (1966) 642. Chorazy, M, Bendich, A, Borenfreund, E & Hutchison, D J, J cell biol 19 (1963) 59. Chorazy, M, Bendich, A, Borenfreund, E, Ittensohn, 0 L & Hutchison, D J, J cell biol 19 (1963) 71. 5. Gartler, S M, Biochem biophys res comm 3 (1960) 127. 6. Huberman, J A & Attardi, G, J cell biol 31 (1966) 95. I. Ittensohn, 0 L & Hutchison, D J, Exptl cell res 55 (1969) 149. 8. Kay, E R M, Trans NY acad sci 28 (1966) 726. 9. Ledoux, L, Progr nucl acid res 4 (1965) 231. 10. Maio, J J & Schildkraut, C L, Methods in cell physiology (ed D M Prescott) vol. 2, p. 113. Academic Press, New York (1966). 11. Robbins. E & Marcus, P I. Science 144 (I 964) 1152. 12. Salzman, N P, Moore, D E & Mendelsohn, J, Proc natl acad sci US 56 (1966) 1449. 13. Terasima, T & Tolmach,‘L J,’ Exptl cell res 30 (1963) 344. 14. Yosida, T H & Sekiguchi, T, Mol and gen genet 103 (1968) 253. Received February 23, 1970