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Ratios of nuclear proteins to DNA for rat and mouse tumors and possible effect of cytoplasmic fibrils in isolating nuclei
Experimental
RATIOS MOUSE
OF NUCLEAR TUMORS
AND
FIBRILS J. MAGLIOZZI,
Cell Research 67 (1971) 111-123
PROTEINS POSSIBLE
TO DNA EFFECT
IN ISOLATING
FOR
RAT
AND
OF CYTOPLASMIC
NUCLEI
D. PURO, CELIA LIN, R. ORTMAN
and A. L. DOUNCE
The University of Rochester, School of Medicine and Dentistry, Biochemistry Department, and Division of Oncology, Rochester, N. Y. 14620, and The City College, Biology Department, New York City, N.Y. 10031, USA
SUMMARY Nuclei isolated from Walker rat carcinoma. one of the Morris hepatomas. and two mouse ascites tumors, using Pb2+ to stabilize the nuclei and Triton N-101 to help remove cytoplasm, showed total histone to DNA ratios ranging from 1.5 to 2.5, and residual protein to DNA ratios of 1.5 to 4.0, with most values falling in the range of 2.0 to 3.0. Tabbed nuclei isolated from Walker tumor in 0.44 M sucrose at DH 3.8 or at higher PH values in the absence of Triton showed masses of fine fibers in the tabs: Similar fibers-were found in electron microscope studies of whole cells from Walker tumor and Ehrlich ascites sarcoma. These fibers may bind parts of the cytoplasm to the nucleus, accounting for difficulties in isolating clean nuclei. In order to carry out successful isolation of tumor cell nuclei, the tumor cells must be harvested from the host animals within a limited period of time, since the cells became increasingly resistant to rupture the longer they remain within the animals.
The relative ease or difficulty that one encounters in isolating nuclei is a function of several factors, the most crucial of which is the degree of difficulty of liberating nuclei from cytoplasm among tissues of different origin. The difficulty is most pronounced in the case of mammalian tumors. Methods routinely used to isolate rat liver nuclei are demonstrably unsatisfactory when applied to tumor tissues such as that of the Walker 256 carcinoma. Recent work in this laboratory has been directed towards improving methods of isolating nuclei of the Walker carcinoma using synthetic detergents, principally Triton
N-101, which seems slightly superior to Triton X-100. The methods to be reported are effective in the pH range of 6.5 to 7.0. The use of such a high pH without loss of nuclear histones is made possible by the addition to the homogenate of heavy metal cations, especially Pb2+, at very low concentrations for stabilizing the nuclei, apparently by reducing autolysis. The presence of the Triton detergent is the crucial factor permitting liberation of clean nuclei from cells within the pH range mentioned, but the effectiveness of the detergent is greatly diminished if the pH is lowered to 5.8-6.0 or 3.8. Walker nuclei isolated by the methods Exptl
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112 J. hfagliozzi et al. described are reasonably free of adherent cytoplasmic tabs, which can be extensive contaminators of nuclei isolated from Walker tumor and other types of tumor cells. Our investigations of tabs indicate that they are probably attached structurally to the tumor cell nucleus; they certainly do not represent loose aggregations of cytoplasmic material adhering to the nucleus. In the work reported in this paper the globulins, histones, DNA and residual protein of the isolated nuclei have been separated and the ratios of each to DNA determined. The effect of varying the concentrations of Pb2+, on the ease of isolating the nuclei has been investigated. The methods described employ aqueous media throughout and follow the traditional plan of homogenization, centrifugation, specific gravity flotation, and washing of the nuclear pellet to achieve acceptable purity. They are outgrowths of earlier attempts to isolate Walker nuclei using CaCI, and sucrose, with and without detergent, which have previously been carried out in this laboratory, as well as of work reported by other investigators, some of which is listed in reference [8]. The work of Busch [5] has emphasized the necessity of isolating Walker nuclei at relatively high pH.
METHODS Transplantation
and preparation
of tissue
(a) Walker carcinoma 256: The Walker 256 carcinoma line was obtained at Roswell Park Cancer Institute in Buffalo, N. Y., courtesy of Dr Fred Rosen. It was transplanted every 2 weeks into Holtzman female albino rats, which at procurement weighed 15&200 g. Inoculation was accomplished by inserting small pieces of the tumor subcutaneously in the side of the rat slightly towards the back and about halfway between the axilla and groin. For best results, the tumors should be harvested 14-21 days after inoculation, before ulceration appears. The rats are decapitated and the tumor is dissected out, cut open, and blood and as much Exptl
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necrotic tissue as possible are removed. The tissue is then forced through a fine mesh stainless steel sieve using an ordinary nestle. An aaueous solution of 0.25 M sucrose poured over the tumor during this treatment facilitates the dissociation of cells. The suspension thus obtained should then be repeatedly filtered through fine mesh gauze. after which the cells are recovered by mild centrifugation (10 min at 500 g). (6) Morris 5123 hepatoma: This tumor was obtained courtesy of Dr Joseph Lee of the Department of Pathology, University of Rochester School of Medicine. Buffalo strain rats were used and the incubation time was 21 days. The procedures for transplantation and tissue processing were the same as for the Walker tumor. (c) TA-3 ascites cell carcinoma: This carcinoma as well as the Ehrlich-Lettre ascites sarcoma was donated by Dr Theodore Hauschka of Roswell Park Memorial Cancer Institute. HAICR Swiss mice. obtained from Roswell Park, were used to carry the cells. The tumor cells were transferred once a week. Transfer was accomplished by the injection of ascites fluid (containing tumor cells) diluted 1 : 100 in mammalian Ringer solution. Immediately prior to isolation, the mice were sacrificed by etherization and neck fracture. The ascites fluid was removed from the peritoneum and the cells were collected by centrifugation at 1 500 rpm. (d) Ehrlich-Lettre ascites tumor cells: The mouse strain used to carry this malignancy was the same as that used for Hauschka TA-3 tumor cells. The procedures for cell transfer and preparation of cells were the same as those used in the case of the Hauschka ascites tumor cells except that cell transfer was made without dilution.
Isolation of nuclei Method using Pb2+ with Triton N-101 : All operations are carried out as close to 0°C as possible. The cellular pellet is diluted approx. 1 to 10, v/v, with the following solution: Sucrose, 0.44 M: neutral Pb(Ac), 0.0002 M; Triton N-101 (Rohm & Hass, Inc.; Philadelphia, Pa) 0.3 % (v/v). (Triton solution No 1). The final homogenate pH should be between 6.5 and 7.0; closer pH regulation is unnecessary. No attempt should ever be made to alter the pH by adding acid or base, since the resultant increase in ionic strength renders the cells less susceotible to breakage. The observed homogenate pH has always been between 6.5 and 7.0. Slightly higher pH might be tolerated; a lower pH cannot be. If this pH has been attained at a dilution sliahtlv lower than 1 to 10, further dilution is avoided. This mixture is shaken and introduced into a Dounce ball-type homogenizer with a loosely-fitting pestle. Five passes of the pestle are sufficient to break most of the cells. At this point at least 90 % of the nuclei should have been liberated from the cells; more homogenization is required using the loose pestle if this is not the case. The homogenate is filtered through Curity no. 60 cheesecloth in a cold room. Squeezing the liquid through the cheesecloth is undesirable, since it enhances contamination of preparation with fiber,
Ratios of nuclear proteins to DNA for rat and mouse tumors
113
1. Analyses of nuclei of Walker tumor and mouse ascites carcinoma TA, for percent DNA and the ratio of the various protein fractions to DNA
Table
Percent DNA and Ratios
Walker tumor nuclei A (3 Runs)
B (5 Runs)
Percent DNA Glob/ DNA
9.19 (7.68-10.2) 0.30 (0.22-0.38) 1.49 (I .26-1.61) 3.52 (2.35-4.15) 5.31 (4.33-5.53)
10.46 (9.00-14.90) 0.21 (0.09-0.41) 1.61 (1.42-1.73) 2.62 (2.06-4.05) 4.45 (3.894.35)
Htl DNA PTi
Ptl DNA
Nuclei from mouse ascites TA, carcinoma C (2 Runs)
A (2 Runs)
B (2 Runs)
C (1 Run)
13.20, 12.50
8.25
17.60
0.48, 0.48
0.33, 0.46
0.14
1.44, 1.60
1.79, 1.78
0.72
1.96, 2.30
2.16, 2.31
1.85
3.91, 4.38
4.28, 4.54
2.71
8.27
10.4, 12.3 0.19, 0.23 0.77, 0.54 2.36, 2.80 3.32, 3.58
Glob, globulin; Ht, histone; P,, residual protein; P,, total protein. A, 0.1 N HCl used to extract histones (with both tumors). B, Histone extracted in 0.2 N HCl. C, No lead used in the isolation.
which occurs in large quantities in solid tumors [l]. The homogenate is then filtered through no. 120 cheesecloth or even finer material. The nuclei are next removed from the homogenate by centrifugation at 1 500 rpm (350 g) for 15 min in the refrigerated International centrifuge No. 9, using rotor No. 284. The pellet obtained by the above centrifugation is then diluted 1: 10 v/v, with the following solution: Sucrose, 0.44 M; MgCl,, 0.001 M; Triton N-101, 0.3 % (Triton solution no. 2). The suspension is next homogenized with three or four passes of the loosely-fitting pestle to suspend the nuclei evenly. Then five passes of the tightlyfitting pestle are carried out. The nuclei are recovered by centrifugation at 1 600 rpm. The pellet is drained of solution for l-2 min and suspended in 5 to 10 vol 2.2 M sucrose. The mixture is homogenized in a previously dried ball-type homogenizer using the tightly fitting pestle until microscopic inspection has indicated that all agglutinated nuclei are thoroughly dissociated. The final pH of the suspension should be as high as possible, preferably between 6.5 and 7.0, to insure a clean separation of nuclei from whole cells. The syrupy suspension is then centrifuged for 10 min at 27 000 rpm in a refrigerated Mode1 L ultracentrifuge using head no. 30 (Chauveau step). This procedure floats whole cells and cytoplasm away from the nuclei, which collect on the outer walls of the centrifuge tube. The nuclei are scraped from the walls of the centrifuge tube and suspended in 3-5 vol of 1 % (w/v) gum arabic, the pH of which has been adjusted to pH 5.8 with 0.1 M NaOH. After 15 min the suspension is centrifuged at 1 000 rpm for 15 min. The nuclei are then resuspended in the gum arabic solution s-
711811
and allowed to stand for an additional 15 min, after which they are recentrifuged at 800 rpm for 15 min. After the gum arabic treatment, the nuclei are suspended in distilled water by gentle swirling. They are collected by gentle centrifugation at about 1 000 rpm (200 g), and are treated once with Triton solution no. 2 (Sucrose, 0.44 M; MgCl,, 0.001 M; Triton N-101, 0.3 %). Centrifugation is then carried out at 1 300 rpm (300 g), for 10 min. The nuclei are finally washed twice in distilled water. If the nuclei still contain cytoplasmic particles, a repetition of the Chauveau step followed by a repetition of the terminal washes may be necessary. In some cases nuclei were isolated as described above except that no lead was used in the homogenate (see Results section and tables 1, 2). In these cases traces of lead were removed from the glassware by washing with cont. HNO,.
Methods for obtaining nuclei with attached cytoplasmic tabsfor electron microscopic work Initially, tabbed Walker nuclei were prepared by homogenizing whole tumor tissue in one volume of 0.44 M sucrose, the pH being adjusted to 3.8 with 0.1 M citric acid. Both loose and tight pestles were used to homogenize the tissue and break as many cells as possible. After homogenization, the pH was checked and readjusted to 3.8 with 0.01 M citric acid. Prior to centrifugation, the homogenate was filtered through two layers of Curity cheesecloth No. 60. The homogenate was diluted 1 :lO with more 0.44 M sucrose and centrifugedat 1 500 rpm (350 g) for 30 min. Exptl
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114 J. Magliozzi et al. Table 2. Analyses fractions to DNA
of nuclei from
Ehrlich-Lettre’
Percent DNA and Ratios
(14 days) (19 days) Prep. la Prep. 2a
mouse ascites sarcoma for ratios of protein
(7 days or younger)
Percent DNA Glob/DNA H,/DNA P,/DNA P,/DNA
13.60 0.28 2.14 3.28 5.70
11.50 0.31 2.59 2.65 5.55
(llday~) .
Prep. 4b
9.44 1.00 3.27 3.21 6.55
9.40 0.30 1.50 2.67 4.56
Prep. 5b 10.40 0.20 1.90 2.50 4.60
Prep. 6b
Average 4 through 6
9.65 0.36 1.44 2.30 4.10
9.82 0.29 1.61 2.49 4.42
Glob, globulin; H,, total histone; P,, residual protein; P,, total protein. a Histones extracted in 0.1 N HCl. b Histones extracted in 0.2 N HCl; nuclei cleaner than in the rest of the cases. 14 days etc. refer to the number of days between inoculation of the tumor and harvesting.
The pellet thus obtained was suspended in 0.44 M sucrose and 0.3 % Triton N-101 by homogenization, using the loose pestle, and the pH was checked and adjusted to 3.8 if necessary with 0.01 M citric acid. The loose pestle was then replaced with the tight one and the suspension was homogenized vigorously. Up to 50 passes were at times necessary to remove granules from the tabs. The pellet was collected by centrifugation at 1 000 rpm (200 g) for 20 min and washed once or twice with distilled Hz0 prior to fixation. Tabbed Walker nuclei were subsequently prepared more easily by homogenizing the tumor in Pb2+ acetate-sucrose solution from which the Triton had been omitted. (Same composition in respect to Pb2+ and sucrose as that of Triton solution no. 1.) Around 20-25 strokes of the loose pestle are sufficient to break about 50 % of the whole cells and give a very high yield of tabbed nuclei. The preparation was filtered once through Curity cheesecloth no. 120 and centrifuged at 1 200 rpm. The pellet was washed once with the detergent-free homogenizing medium, and a 1 ml aliquot was withdrawn, sedimented to a tight pellet at room temperature, and fixed for electron microscopy. Microscopic examination after homogenizing Ehrlich cells that had been allowed to remain in the host animals for 2 weeks showed the presence of whole cells and cell remnants but very few nuclei. These whole cells and cell remnants were recovered by centrifugation and were fixed for microscopy, after it became clear that the use of the sucrose-lead acetate-Triton homogenizing solution was totally unsuccessful in attempts to rupture these cells.
Methods of electron microscopy A 1 ml aliquot of a nuclear or cellular suspension was spun for 7000 g for 3 min into a tight pellet. Five ml of Karnovsky glutaraldehyde-formaldehyde fixative [17] was layered on top of the pellet and left for 90 min at room temperature. Other primary fixatives used were glutaraldehyde 2.0 %, sucrose Exptl
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67
0.25 M, MgCl, 0.001 M, and 10 % acrolein in phosphate buffer. Post-fixation in 1 % 0~0, buffered in Verona1 was carried out in for 60 or 90 min at 0°C or room temperature. The specimen was rapidly dehydrated in graded ethanol and absolute propylene oxide, and embedded in Araldite or Epon-Araldite. Sections were cut on the LKB Ultrotome and were stained for 30 min in uranyl acetate and for 5 min in Reynolds lead citrate. Grids were examined on the RCA EMU-3 electron microscope with an objective aperture of 50 pm and a beam voltage of 50 or 100 kV.
Methods for protein extraction and DNA determination The solvents and methods used to extract the globulins, histones, residual proteins and DNA are discussed in ref. [9, 241. DNA was determined by the Dische-Schneider method [24] both from an aliquot of whole nuclei and from the DNA-protein residue left after extraction of histones, globulins, and lipids. Globulins were precipitated by heat denaturation, dried and weighed. Most of the histone was precipitated with NH,OH at pH 11. A small portion of the total histone is however soluble in NH,OH and must be precipitated by the addition of 3 vol of ethanol. Both histone fractions were dried at 105°C and weighed. The insoluble residual protein that remained after extraction of nucleic acid with hot 5 % trichloroacetic acid and washing with ethanol was also dried at 105°C and weighed.
RESULTS Qualitative observations Phase microscope photographs of nuclei isolated from Walker carcinoma 256, Hauschka ascites carcinoma TA-3, Ehrlich ascites sar-
Ratios of nuclear proteins to DNA for rat and mouse tumors
115
Figs 1, 2. The lacunae in some of the nuclei do not indicate loss of material from nuclei during the isolation procedure, but rather result from difficulties with fixation and probably embedding. The electron microscope photos of the nuclei have been included mainly to show loss of the outer nuclear membrane. No attempt is intended to show nuclear fine structure. Fig. 1. (a) Nuclei isolated from Walker carcinoma 256. Phase contrast, ca x 750. (b) Nuclei isolated from Hauschka ascites carcinoma TA-3. Phase contrast,, ca x 750. (c) Nuclei isolated from Ehrlich-Lettre ascites sarcoma. Phase contrast, ca x 750. (d) Nuclei isolated from Morris hepatoma. Phase contrast, ca x 750. Exptl
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116 J. Magliozzi et al. Table 3. Analyses of nuclei isolatedfrom Morris hepatomafor ratios of protein fractions to DNA Percent DNA and ratios Percent DNA Glob/DNA H,/DNA P,/DNA P,/DNA
Prep. la 13.10 0.25 2.00 1.43 3.68
Prep. 2b 10.60 0.33 1.85 1.95 4.12
Ave. 11.90 0.29 1.93 1.69 3.85
Glob, globulin; Ht, total histone; P,, residual protein; P,, total protein. a 0.1 N HCI used to extract histones. b 0.2 N HCl used to extract histones.
Fig. 2. Electron micrographs of nuclei from: (a) Walker carcinoma 256; (b) Hauschka ascites carcinoma TA,; (c) Ehrlich-Lettre ascites sarcoma. Fixation: (a, b) Karnovsky fixative, pH 7.22; (c) 2 % glutaraldehyde0.001 M MgCl,. Staining: (a, b, c) uranyl acetate, lead citrate. Magnification (a) x 3 400; (b) x 1 800; (c) xl 300.
coma, and Morris hepatoma are shown in fig. la-d respectively. These photographs refer to small nuclear aliquots withdrawn from preparations and fixed immediately after they were finished. Some of the nuclei still retain tabs or thin shells of Exptl
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cytoplasm which have lost visible particulate matter and therefore probably the dissolved part of the cytoplasm at least. In one or two casescleaner nuclei than are shown were obtained from the Morris hepatoma when the cells were harvested between 1 and 1.5 weeks after inoculation. Electron micrographs of nuclei from Walker carcinoma 256, Hauschka ascitescarcinoma TA, and Ehrlich-LettrC ascites carcinoma, respectively are shown in fig. 2a- c. It can be seen that the outer nuclear membrane has been lost, presumably because of the use of the Triton N-101 in the isolation procedure. A similar observation has been recorded by Zalta et al. [15]. The same thing has been observed subsequently by Berkowitz et al. [3]. Loss of the outer nuclear membrane and the lipid associated with the inner membrane might account for the observed failure of the nuclear pellet to blacken markedly in buffered osmic acid. An electron micrograph of a tabbed Walker carcinoma nucleus is shown in fig. 3. A mass of fiber can be seen in the tab, part of which (the ropelike structure) seemsto be attached to the nuclear membrane. Most of the tabbed Walker tumor nuclei examined that had been fixed in Karnovsky fixative
Ratios of nuclear proteins to DNA for rat and mouse tumors
117
Fig. 3. Electron micrograph of tabbed Walker carcinoma nucleus (see Methods section). Fixation: Karnovsky fixative, pH 7.22. Staining: Uranyl acetate, lead citrate. Magnification ca x 53 500.
showed such fibers. Dr Keith Porter, who examined some of our early electron micrographs, stated that in his opinion the fibers
represented distorted microtubules from the centrosphere, a region around the centriole. (See [l, 7, 221 for a description of microExptl
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118
J. Magliozzi et al.
Fig. 4. Part of whole cell from Walker tumor showing microfibers. Silver section. Fixation: 10 % acrolein in pH 7.3 phosphate buffer. Post fixation: Palade 1 % osmic acid for 1.5 h. Araldite embedding. Staining: Uranyl acetate, lead citrate. Magnification x 69 000. We are indebted to Mr J. van Blerkom of the University of Colorado who took this electron micrograph using a Philips 200 instrument.
Exptl
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Ratios of nuclear proteins to DNA for rat and mouse tumors Table 4. Analyses for ratios of protein fractions to DNA of nuclei isolated from normal rat liver by the lead-Triton method developed for isolating cancer cell nuclei Percent DNA and ratios
Prep. 1
Prep. 2
Ave.
Per cent DNA Glob/DNA HJDNA P,/DNA PJDNA
8.50 0.17 2.64 2.48 5.28
9.40 0.20 1.78 2.66 4.61
8.95 0.19 2.16 2.57 4.95
Glob, globulin; H,, total histone; P,, residual protein; P,, total protein.
tubules). Ehrlich ascites sarcoma nuclei bearing tabs, or cells surrounded by shells of cytoplasm (cell remnants) showed similar fibers and at times endoplasmic reticulum, mitochondria, and inclusion bodies. The same type of fibers was seen in whole cells of the Walker tumor (seefig. 4). Apparently similar fibers have been reported by Sykes et al. [23]. The Ehrlich sarcoma cell remnants used in this work were obtained from Ehrlich ascites sarcoma cells that had been allowed to remain in the host mice for 2 weeks before harvesting (seeExperimental section). The method described in this paper for isolating the malignant nuclei does not yield nuclei capable of forming gels [19], indicating that there has been some kind of partial autolysis of the macromolecules of the chromatin during the isolation procedure. We believe on the basis of preliminary evidence that this attack was made by DNAase; the lead used in the isolation acts as a protease inhibitor. Whatever autolysis occurs apparently does not, however, appreciably change the ratios of the principal protein fractions to DNA, since a control experiment showed that normal rat liver nuclei isolated by the same method as was used for isolating the tumor nuclei still show the usual ratios for
119
the various protein fractions to DNA that are characteristic of rat liver nuclei that are capable of gel formation (see table 4). The rat liver nuclei isolated by the method for isolating tumor nuclei, like the latter, do not form gels. It has been found possible to obtain liver cell nuclei capable of forming gels by use of the same method as described for the isolation of the cancer cell nuclei, except for the addition of 0.0002 M indium trichloride in the homogenization and 0.0001 M indium trichloride in the subsequentsteps, where the concentration of Pb2+ was reduced to 0.0001 M. However we have not yet found concentrations of indium and lead that will allow good cell breakage in working with cancer cell nuclei. Analyses of the isolated nuclei for the ratios of the principal protein fractions to DNA The ratios of the “globulin” fraction, the total histone, and the residual protein to DNA for the four tumors studied are shown in tables 1 through 3. As a control the same kind of data is given in table 4 for normal rat liver nuclei isolated by the same method that was used for the cancer cell nuclei. It can be seen that the ratio of total histone to DNA in all casesis considerably above 1, and is close to 2 in the case of the Ehrlich ascites tumor and the Morris hepatoma. The ratio of residual protein to DNA is often in the neighborhood of 3 but is occasionally higher; in the case of the Morris hepatoma, it is slightly under 2, in spite of the fact that owing to the presence of some fiber in these nuclei, one might have expected this ratio to be high rather than low. It is of interest to compare these ratios with corresponding ratios for liver cell nuclei [lo]. If Pb2+ is omitted from the preparation nuclei can be isolated from the Walker & Exptl
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120 J. Magliozzi et al. Hauschka TA3 carcinomas, are greatly reduced and the histone to DNA are lowered than 1.0, as is shown by the 1 and 2.
but the yields ratios of total to values less data in tables
DISCUSSION For a number of years, it was generally held that the problem of breaking cells prior to isolating nuclei could be solved by applying mechanical forces to cells. The investigations of Busch on the relationship between homogenizer pestle clearance and yield of nuclei showed that in the case of the Walker carcinoma, the optimal clearance was about 77 P 151. It is possible to employ non-ionic detergents to aid in the breakage of the tumor cell. Zalta et al. [15] appears to have been the first to use such detergents in isolating cell nuclei. When used at the pH range of 6.57.0, these detergents allow one to break the Walker cells with high efficiency, using the loose pestle. Examples of the detergents suitable for this purpose are Triton X-100 and Triton N-101. Hymer & Kuff [16] introduced the former product in a method suitable for the preparation of rat liver nuclei and mouse plasma cell nuclei; Lazarus & Sporn employed the latter in a method designed to isolate nuclei from the mouse Ehrlich-LettrC sarcoma [ 181. Triton N-101 has recently been used in the isolation of HeLa cell nuclei [3, 201, and non-ionic detergent has come to be accepted quite generally as an aid in isolating tumor cell nuclei. A mixture of ionic and non-ionic detergent has also been used [13]. Either of the above-mentioned detergents causes a sufficient attack on the cytoplasmic matrix so that it can be removed from nuclei by homogenization. A disadvantage in the use of the Tritons is that they appear to destroy mitochondria and lysosomes during Exptl
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homogenization, a process which releases enzymes which attack macromolecules of chromatin and thus render tumor nuclei non-gelable. In this respect, isolation of nuclei in the presence of non-ionic detergent is similar to isolation using water as homogenizing medium. Also, the outer nuclear membrane is removed by the Tritons [3, 161. In spite of these disadvantages, the use of detergent is the only means available at present for obtaining satisfactory removal of cytoplasm from most types of cancer cell nuclei. Although enough cancer cells can be broken in the pH range of 6.5 to 7.0 to liberate most of the nuclei, lowering the pH to 5.8 greatly increases the resistance of the cells to breakage so that satisfactory isolation of nuclei cannot be accomplished, even when detergents are present. Since the use of detergent favors the release of enzymes which tend to attack or destroy nuclei, the introduction of some agent which can suppress these enzymes in order to stabilize the nuclei and prevent loss of nuclear macromolecules is highly desirable and in some cases at least probably necessary if nuclear isolation is to be achieved. In the past, Ca2+ and Mg2+ have often been used for stabilization; these cations probably are effective because of causing condensation of chromatin. In the absence of other enzyme inhibitors however, Ca2+ and Mg2+ cause a loss of the easily extractable histone fraction from liver cell nuclei [lo]. They also tend to enhance the resistance of the cells to breakage. Our recent discovery that Pb2+, Cd2+ and In2+ aid in stabilizing nuclei in cellular homogenates [ll], presumably by inhibiting the action of proteases and other enzymes, has induced us to investigate the use of these metal cations in isolating Walker tumor nuclei. It has been found that Pb2+ can be used in the homogenizing medium in the pH range of 6.5-7.0; the optimal concentra-
Ratios of nuclear proteins to DNA for rat and mouse tumors
tion is 0.0002 M. Pb2+ appears to be the best of the three cations just mentioned in the case of tumor cell nuclei; the use of Cd2+ at 0.0002 M concentration permits liberation of the nuclei but leaves many nuclei with attached cytoplasmic tabs. Indium trichloride is effective as a stabilizer but increases the difficulty of liberating the nuclei. Pb2+ has therefore been used for the most part in this study. It has been found that the use of MgCl, in washing the cancer cell nuclei which have been partially isolated in the presence of Pb2+ is very helpful and probably necessary. Its use throws the nuclei into a more condensed state and enables one to use the tightly-fitting pestle of homogenizer, which is essential for removing residual cytoplasmic tabs from the nuclei. The use of Mg2+ in washing the nuclei also reduces cytoplasmic agglutination, which is increased in washing the nuclei in the presence of lead. It has been determined that the use of Mg 2+ in washing liver cell nuclei subsequent to the use of Pb2+ does not cause loss of the easily extractable histone fraction or otherwise affect the ratios of the principal protein fractions to DNA and therefore its use in the isolation of tumor nuclei subsequent to the use of Pb2+ appears justified. Since the total histone to DNA ratio of about 1.6 (see table 1) found in this study for Walker tumor nuclei lies between the ratio of about 1.Ofound thus far for calf thymus nuclei and the ratio of 2-2.5 found for rat liver nuclei [I 1, 121, it might be thought that sufficient stromal nuclei having an HJDNA ratio of 2.5 were present along with carcinoma nuclei having a ratio of 1.0 to account for the observed intermediate ratio of the nuclear preparation. However this interpretation would require the presence of at least 40 y, stromal cells; microscopic observation indicates that the percentage of stromal cells in
121
the preparation must be far less than this, and hence we ascribe the observed ratio of 1.6 to the tumor cell nuclei themselves. The intermediate ratio for the Hauschka ascites carcinoma nuclei apparently must also be ascribable to the malignant cell nuclei, since no stromal cell nuclei should be present in the nuclei isolated from this malignancy. The lower ratios reported previously for tumor cell nuclei [25] were undoubtedly due to the use of Mg2+ without heavy metal in the isolation procedure. Disc gel electrophoresis of the total histone fractions from the cancer cell nuclei has indicated that probably not more than lo15 % of the fractions are non-histone protein. The use of 0.2 N HCl instead of 0.1 N HCl for extracting the total histone of the tumor nuclei did not on the average result in more than a very small increase in the ratio of total histone to DNA. The difficulty in removing tabs of cytoplasm from cancer cell nuclei is the rule rather than the exception [8]. We know of no explanation for this difficulty that has been offered previously. The ability of the attached tabs to keep their shape in spite of breakage of the cell membrane leads to the deduction that the tabs must possess some sort of framework structure that maintains their shape and keeps them bound to the nuclei. Such a framework structure must be at least in part constructed of protein, since the tab structure was found to be destroyed by pepsin [8]. The presence of fibers in malignant cells has been noted by Sykes et al. [23], who credit Haguenau [14] as being the first to report them. The microfibrils of Haguenau were found in myoepithelial cells and do not appear to be related to the microfibrils discussed by us. The term “tonofibrils” was used in designating these microfibrils which were thought to be characteristic of malignancy. Exptl
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122 J. Magliozzi et al. The fibers found by us in nuclear tabs and cytoplasm of the Walker tumor and Ehrlich ascites sarcoma cells appear similar to those found by Sykes et al. as far as can be judged by the electron micrographs. It seems reasonable to suppose that the fibers found in the tabs and cytoplasm of the Walker tumor and the Ehrlich ascites sarcoma constitute the main part of the framework structure just discussed. Apparently removal of the outer nuclear membrane at least is generally necessary if clean tumor nuclei are to be isolated. This leads one to suspect attachment of the fibers to the outer nuclear membrane. The latter, as well as most of the inner membrane, can be removed by strong citric acid [4] as well as by the use of detergents, and the use of either of these reagents can lead to the isolation of relatively clean nuclei. Another factor that possibly could contribute to the difficulty of isolating nuclei from malignant cells from tissue culture is the presence of intercellular bridges [2]. Busch [6] has reported a method for the isolation of clean nuclei from certain Morris hepatomas which does not involve the use of non-ionic detergent, but this method did not work with all Morris hepatomas. In the same article Busch has criticized the use of osmotic
swelling
followed
by homogeniza-
tion in the presence of detergents in isolating tumor nuclei, on the basis of a report by Penman [21] that nuclei isolated in this way lose completely their 18S RNA. We have not been concerned with nuclear RNA in this work, and moreover we have avoided the use of osmotic swelling and have added a stabilizing agent (Pb2+) that has been shown to protect cell nuclei against proteolytic action. A final point worth emphasizing is the variability in tumors which affects the ease of isolating nuclei in an important manner. If Exptl
Cell Res 67
the tumors grow very slowly and therefore are left in the host animals for some time before being harvested, the isolation of nuclei may be difficult or impossible. This has been noted in the case of the Walker tumor, the Ehrlich ascites tumor, and probably the Morris hepatoma. The situation is particularly interesting in the case of the Ehrlich ascites sarcoma; it is impossible to isolate nuclei by the method described in this paper if the cancer cells are harvested 2 weeks after inoculation, whereas fairly good nuclei can be obtained if the cancer cells are harvested one week after inoculation. It is thus of great importance to make every effort to arrange conditions so as to obtain as suitable tumor or cancer cells as possible if cell nuclei are to be isolated. J. M. gratefully acknowledges support of USPHS Training Grant AM 0100404. R. 0. is indebted to USPHS (5Tl-HD-00133-03) for support of his part in this research. A. L. D. is glad to acknowledge support from the USPHS, National Cancer Institute, grant CA 00994-19.
D. P. is glad to acknowledge support of National Cancer Institute grant CA-1 19801.
REFERENCES 1. Adelman, M R, Borisy, G G, Shelanski, M L, Weisenberg, R C & Taylor, E W, Fed proc 27 (1968) 1186. 2. Bendich, A, Vizoso, A D & Harris, R G, Proc natl acad sci US 54 (1967) 1029. 3. Berkowitz, D M, Kakefuda, T & Sporn, M B, J cell bio142 (1969) 851. 4. Borneus, M, Compt rend acad sci ser D 266 (1968) 596. 5. Busch, H, Starbuck, W C & Davis, J R, Cancer res 19 (1959) 684. 6. Busch, H, Methods in cancer res (ed H Busch) ~014, chap 6, p. 179. Academic Press, New York (1967-1968). 7. Claude, A, Compt rend 254 (1961) 2251. 8. Dounce, A L, Exptl cell res, suppl. 9 (1963) 126. 9. - Nucleic acids (ed E Chargaff & J N Davidson) vol. 2, p. 110. Academic Press, New York (1955). 10. Dounce, A L & Ickowicz, R, Arch biochem biophys 131 (1970) 359. 11. - Ibid 137 (1970) 143. 12. Dounce, A L, Seaman, F & Mackay, M, Arch biochem biophys 117 (1966) 550.
Ratios of nuclear proteins to DNA for rat and mouse tumors 13. Ginzburg-Teitz, Y, Kaufmann, E & Traub, A, Biochim biophys acta 134 (1967) 211. 14. Haguenau, F, Compt rend acad sci 249 (1959) 182. 15. Hubert, M T, Farard, P, Carasso, N, Rozencwajg, R & Zalta, J P, J microscop 1 (1962) 435. 16. Hymer, W- C & Kuff, E L,-J histochem cytochem 12 (1964) 359. 17. Karnovskv. M J. J cell bio127 (1965) 137n. 18. Lazarus, H M & Spom, M B, Proc natl acad sci US 57 (1967) 1386. 19. Mackay, M, Hilgartner, C A & Dounce, A L, Exptl cell res 49 (1968) 533. 20. Munro, G F, Dounce, A L & Lerman, S, Cancer res 55 (1969) 46. 21. Vesco, C & Penman, S, Biochim biophys acta 169 (1968) 188.
123
22. Porter, K R, Principles of biomolecular organisation. Ciba foundation symposium (ed G E W Wolstenholme & N O’Connor) p. 308. Little, Brown & Co, Boston (1966). 23. Sykes, J A, Recher, L, Jernstrom, P H & Whitescarver, J, J natl cancer inst 40 (1968) 195. 24. Umafia, R & Dounce, A L, Exptl cell res 35 (1964) 277. 25. Umafia, R, Updike, S, Randall, J & Dounce, A L, The nucleohistones (ed J Bonner & P Ts’o) p. 200. Holden-Day, San Francisco (1964).
Received December 17 1970 Revised version received March 2, 1971
ExptI Cell Res 67
RATIOS MOUSE
OF NUCLEAR TUMORS
AND
FIBRILS J. MAGLIOZZI,
Cell Research 67 (1971) 111-123
PROTEINS POSSIBLE
TO DNA EFFECT
IN ISOLATING
FOR
RAT
AND
OF CYTOPLASMIC
NUCLEI
D. PURO, CELIA LIN, R. ORTMAN
and A. L. DOUNCE
The University of Rochester, School of Medicine and Dentistry, Biochemistry Department, and Division of Oncology, Rochester, N. Y. 14620, and The City College, Biology Department, New York City, N.Y. 10031, USA
SUMMARY Nuclei isolated from Walker rat carcinoma. one of the Morris hepatomas. and two mouse ascites tumors, using Pb2+ to stabilize the nuclei and Triton N-101 to help remove cytoplasm, showed total histone to DNA ratios ranging from 1.5 to 2.5, and residual protein to DNA ratios of 1.5 to 4.0, with most values falling in the range of 2.0 to 3.0. Tabbed nuclei isolated from Walker tumor in 0.44 M sucrose at DH 3.8 or at higher PH values in the absence of Triton showed masses of fine fibers in the tabs: Similar fibers-were found in electron microscope studies of whole cells from Walker tumor and Ehrlich ascites sarcoma. These fibers may bind parts of the cytoplasm to the nucleus, accounting for difficulties in isolating clean nuclei. In order to carry out successful isolation of tumor cell nuclei, the tumor cells must be harvested from the host animals within a limited period of time, since the cells became increasingly resistant to rupture the longer they remain within the animals.
The relative ease or difficulty that one encounters in isolating nuclei is a function of several factors, the most crucial of which is the degree of difficulty of liberating nuclei from cytoplasm among tissues of different origin. The difficulty is most pronounced in the case of mammalian tumors. Methods routinely used to isolate rat liver nuclei are demonstrably unsatisfactory when applied to tumor tissues such as that of the Walker 256 carcinoma. Recent work in this laboratory has been directed towards improving methods of isolating nuclei of the Walker carcinoma using synthetic detergents, principally Triton
N-101, which seems slightly superior to Triton X-100. The methods to be reported are effective in the pH range of 6.5 to 7.0. The use of such a high pH without loss of nuclear histones is made possible by the addition to the homogenate of heavy metal cations, especially Pb2+, at very low concentrations for stabilizing the nuclei, apparently by reducing autolysis. The presence of the Triton detergent is the crucial factor permitting liberation of clean nuclei from cells within the pH range mentioned, but the effectiveness of the detergent is greatly diminished if the pH is lowered to 5.8-6.0 or 3.8. Walker nuclei isolated by the methods Exptl
Cell Res 67
112 J. hfagliozzi et al. described are reasonably free of adherent cytoplasmic tabs, which can be extensive contaminators of nuclei isolated from Walker tumor and other types of tumor cells. Our investigations of tabs indicate that they are probably attached structurally to the tumor cell nucleus; they certainly do not represent loose aggregations of cytoplasmic material adhering to the nucleus. In the work reported in this paper the globulins, histones, DNA and residual protein of the isolated nuclei have been separated and the ratios of each to DNA determined. The effect of varying the concentrations of Pb2+, on the ease of isolating the nuclei has been investigated. The methods described employ aqueous media throughout and follow the traditional plan of homogenization, centrifugation, specific gravity flotation, and washing of the nuclear pellet to achieve acceptable purity. They are outgrowths of earlier attempts to isolate Walker nuclei using CaCI, and sucrose, with and without detergent, which have previously been carried out in this laboratory, as well as of work reported by other investigators, some of which is listed in reference [8]. The work of Busch [5] has emphasized the necessity of isolating Walker nuclei at relatively high pH.
METHODS Transplantation
and preparation
of tissue
(a) Walker carcinoma 256: The Walker 256 carcinoma line was obtained at Roswell Park Cancer Institute in Buffalo, N. Y., courtesy of Dr Fred Rosen. It was transplanted every 2 weeks into Holtzman female albino rats, which at procurement weighed 15&200 g. Inoculation was accomplished by inserting small pieces of the tumor subcutaneously in the side of the rat slightly towards the back and about halfway between the axilla and groin. For best results, the tumors should be harvested 14-21 days after inoculation, before ulceration appears. The rats are decapitated and the tumor is dissected out, cut open, and blood and as much Exptl
Cell Res 67
necrotic tissue as possible are removed. The tissue is then forced through a fine mesh stainless steel sieve using an ordinary nestle. An aaueous solution of 0.25 M sucrose poured over the tumor during this treatment facilitates the dissociation of cells. The suspension thus obtained should then be repeatedly filtered through fine mesh gauze. after which the cells are recovered by mild centrifugation (10 min at 500 g). (6) Morris 5123 hepatoma: This tumor was obtained courtesy of Dr Joseph Lee of the Department of Pathology, University of Rochester School of Medicine. Buffalo strain rats were used and the incubation time was 21 days. The procedures for transplantation and tissue processing were the same as for the Walker tumor. (c) TA-3 ascites cell carcinoma: This carcinoma as well as the Ehrlich-Lettre ascites sarcoma was donated by Dr Theodore Hauschka of Roswell Park Memorial Cancer Institute. HAICR Swiss mice. obtained from Roswell Park, were used to carry the cells. The tumor cells were transferred once a week. Transfer was accomplished by the injection of ascites fluid (containing tumor cells) diluted 1 : 100 in mammalian Ringer solution. Immediately prior to isolation, the mice were sacrificed by etherization and neck fracture. The ascites fluid was removed from the peritoneum and the cells were collected by centrifugation at 1 500 rpm. (d) Ehrlich-Lettre ascites tumor cells: The mouse strain used to carry this malignancy was the same as that used for Hauschka TA-3 tumor cells. The procedures for cell transfer and preparation of cells were the same as those used in the case of the Hauschka ascites tumor cells except that cell transfer was made without dilution.
Isolation of nuclei Method using Pb2+ with Triton N-101 : All operations are carried out as close to 0°C as possible. The cellular pellet is diluted approx. 1 to 10, v/v, with the following solution: Sucrose, 0.44 M: neutral Pb(Ac), 0.0002 M; Triton N-101 (Rohm & Hass, Inc.; Philadelphia, Pa) 0.3 % (v/v). (Triton solution No 1). The final homogenate pH should be between 6.5 and 7.0; closer pH regulation is unnecessary. No attempt should ever be made to alter the pH by adding acid or base, since the resultant increase in ionic strength renders the cells less susceotible to breakage. The observed homogenate pH has always been between 6.5 and 7.0. Slightly higher pH might be tolerated; a lower pH cannot be. If this pH has been attained at a dilution sliahtlv lower than 1 to 10, further dilution is avoided. This mixture is shaken and introduced into a Dounce ball-type homogenizer with a loosely-fitting pestle. Five passes of the pestle are sufficient to break most of the cells. At this point at least 90 % of the nuclei should have been liberated from the cells; more homogenization is required using the loose pestle if this is not the case. The homogenate is filtered through Curity no. 60 cheesecloth in a cold room. Squeezing the liquid through the cheesecloth is undesirable, since it enhances contamination of preparation with fiber,
Ratios of nuclear proteins to DNA for rat and mouse tumors
113
1. Analyses of nuclei of Walker tumor and mouse ascites carcinoma TA, for percent DNA and the ratio of the various protein fractions to DNA
Table
Percent DNA and Ratios
Walker tumor nuclei A (3 Runs)
B (5 Runs)
Percent DNA Glob/ DNA
9.19 (7.68-10.2) 0.30 (0.22-0.38) 1.49 (I .26-1.61) 3.52 (2.35-4.15) 5.31 (4.33-5.53)
10.46 (9.00-14.90) 0.21 (0.09-0.41) 1.61 (1.42-1.73) 2.62 (2.06-4.05) 4.45 (3.894.35)
Htl DNA PTi
Ptl DNA
Nuclei from mouse ascites TA, carcinoma C (2 Runs)
A (2 Runs)
B (2 Runs)
C (1 Run)
13.20, 12.50
8.25
17.60
0.48, 0.48
0.33, 0.46
0.14
1.44, 1.60
1.79, 1.78
0.72
1.96, 2.30
2.16, 2.31
1.85
3.91, 4.38
4.28, 4.54
2.71
8.27
10.4, 12.3 0.19, 0.23 0.77, 0.54 2.36, 2.80 3.32, 3.58
Glob, globulin; Ht, histone; P,, residual protein; P,, total protein. A, 0.1 N HCl used to extract histones (with both tumors). B, Histone extracted in 0.2 N HCl. C, No lead used in the isolation.
which occurs in large quantities in solid tumors [l]. The homogenate is then filtered through no. 120 cheesecloth or even finer material. The nuclei are next removed from the homogenate by centrifugation at 1 500 rpm (350 g) for 15 min in the refrigerated International centrifuge No. 9, using rotor No. 284. The pellet obtained by the above centrifugation is then diluted 1: 10 v/v, with the following solution: Sucrose, 0.44 M; MgCl,, 0.001 M; Triton N-101, 0.3 % (Triton solution no. 2). The suspension is next homogenized with three or four passes of the loosely-fitting pestle to suspend the nuclei evenly. Then five passes of the tightlyfitting pestle are carried out. The nuclei are recovered by centrifugation at 1 600 rpm. The pellet is drained of solution for l-2 min and suspended in 5 to 10 vol 2.2 M sucrose. The mixture is homogenized in a previously dried ball-type homogenizer using the tightly fitting pestle until microscopic inspection has indicated that all agglutinated nuclei are thoroughly dissociated. The final pH of the suspension should be as high as possible, preferably between 6.5 and 7.0, to insure a clean separation of nuclei from whole cells. The syrupy suspension is then centrifuged for 10 min at 27 000 rpm in a refrigerated Mode1 L ultracentrifuge using head no. 30 (Chauveau step). This procedure floats whole cells and cytoplasm away from the nuclei, which collect on the outer walls of the centrifuge tube. The nuclei are scraped from the walls of the centrifuge tube and suspended in 3-5 vol of 1 % (w/v) gum arabic, the pH of which has been adjusted to pH 5.8 with 0.1 M NaOH. After 15 min the suspension is centrifuged at 1 000 rpm for 15 min. The nuclei are then resuspended in the gum arabic solution s-
711811
and allowed to stand for an additional 15 min, after which they are recentrifuged at 800 rpm for 15 min. After the gum arabic treatment, the nuclei are suspended in distilled water by gentle swirling. They are collected by gentle centrifugation at about 1 000 rpm (200 g), and are treated once with Triton solution no. 2 (Sucrose, 0.44 M; MgCl,, 0.001 M; Triton N-101, 0.3 %). Centrifugation is then carried out at 1 300 rpm (300 g), for 10 min. The nuclei are finally washed twice in distilled water. If the nuclei still contain cytoplasmic particles, a repetition of the Chauveau step followed by a repetition of the terminal washes may be necessary. In some cases nuclei were isolated as described above except that no lead was used in the homogenate (see Results section and tables 1, 2). In these cases traces of lead were removed from the glassware by washing with cont. HNO,.
Methods for obtaining nuclei with attached cytoplasmic tabsfor electron microscopic work Initially, tabbed Walker nuclei were prepared by homogenizing whole tumor tissue in one volume of 0.44 M sucrose, the pH being adjusted to 3.8 with 0.1 M citric acid. Both loose and tight pestles were used to homogenize the tissue and break as many cells as possible. After homogenization, the pH was checked and readjusted to 3.8 with 0.01 M citric acid. Prior to centrifugation, the homogenate was filtered through two layers of Curity cheesecloth No. 60. The homogenate was diluted 1 :lO with more 0.44 M sucrose and centrifugedat 1 500 rpm (350 g) for 30 min. Exptl
Cell
Res 67
114 J. Magliozzi et al. Table 2. Analyses fractions to DNA
of nuclei from
Ehrlich-Lettre’
Percent DNA and Ratios
(14 days) (19 days) Prep. la Prep. 2a
mouse ascites sarcoma for ratios of protein
(7 days or younger)
Percent DNA Glob/DNA H,/DNA P,/DNA P,/DNA
13.60 0.28 2.14 3.28 5.70
11.50 0.31 2.59 2.65 5.55
(llday~) .
Prep. 4b
9.44 1.00 3.27 3.21 6.55
9.40 0.30 1.50 2.67 4.56
Prep. 5b 10.40 0.20 1.90 2.50 4.60
Prep. 6b
Average 4 through 6
9.65 0.36 1.44 2.30 4.10
9.82 0.29 1.61 2.49 4.42
Glob, globulin; H,, total histone; P,, residual protein; P,, total protein. a Histones extracted in 0.1 N HCl. b Histones extracted in 0.2 N HCl; nuclei cleaner than in the rest of the cases. 14 days etc. refer to the number of days between inoculation of the tumor and harvesting.
The pellet thus obtained was suspended in 0.44 M sucrose and 0.3 % Triton N-101 by homogenization, using the loose pestle, and the pH was checked and adjusted to 3.8 if necessary with 0.01 M citric acid. The loose pestle was then replaced with the tight one and the suspension was homogenized vigorously. Up to 50 passes were at times necessary to remove granules from the tabs. The pellet was collected by centrifugation at 1 000 rpm (200 g) for 20 min and washed once or twice with distilled Hz0 prior to fixation. Tabbed Walker nuclei were subsequently prepared more easily by homogenizing the tumor in Pb2+ acetate-sucrose solution from which the Triton had been omitted. (Same composition in respect to Pb2+ and sucrose as that of Triton solution no. 1.) Around 20-25 strokes of the loose pestle are sufficient to break about 50 % of the whole cells and give a very high yield of tabbed nuclei. The preparation was filtered once through Curity cheesecloth no. 120 and centrifuged at 1 200 rpm. The pellet was washed once with the detergent-free homogenizing medium, and a 1 ml aliquot was withdrawn, sedimented to a tight pellet at room temperature, and fixed for electron microscopy. Microscopic examination after homogenizing Ehrlich cells that had been allowed to remain in the host animals for 2 weeks showed the presence of whole cells and cell remnants but very few nuclei. These whole cells and cell remnants were recovered by centrifugation and were fixed for microscopy, after it became clear that the use of the sucrose-lead acetate-Triton homogenizing solution was totally unsuccessful in attempts to rupture these cells.
Methods of electron microscopy A 1 ml aliquot of a nuclear or cellular suspension was spun for 7000 g for 3 min into a tight pellet. Five ml of Karnovsky glutaraldehyde-formaldehyde fixative [17] was layered on top of the pellet and left for 90 min at room temperature. Other primary fixatives used were glutaraldehyde 2.0 %, sucrose Exptl
Cell
Res
67
0.25 M, MgCl, 0.001 M, and 10 % acrolein in phosphate buffer. Post-fixation in 1 % 0~0, buffered in Verona1 was carried out in for 60 or 90 min at 0°C or room temperature. The specimen was rapidly dehydrated in graded ethanol and absolute propylene oxide, and embedded in Araldite or Epon-Araldite. Sections were cut on the LKB Ultrotome and were stained for 30 min in uranyl acetate and for 5 min in Reynolds lead citrate. Grids were examined on the RCA EMU-3 electron microscope with an objective aperture of 50 pm and a beam voltage of 50 or 100 kV.
Methods for protein extraction and DNA determination The solvents and methods used to extract the globulins, histones, residual proteins and DNA are discussed in ref. [9, 241. DNA was determined by the Dische-Schneider method [24] both from an aliquot of whole nuclei and from the DNA-protein residue left after extraction of histones, globulins, and lipids. Globulins were precipitated by heat denaturation, dried and weighed. Most of the histone was precipitated with NH,OH at pH 11. A small portion of the total histone is however soluble in NH,OH and must be precipitated by the addition of 3 vol of ethanol. Both histone fractions were dried at 105°C and weighed. The insoluble residual protein that remained after extraction of nucleic acid with hot 5 % trichloroacetic acid and washing with ethanol was also dried at 105°C and weighed.
RESULTS Qualitative observations Phase microscope photographs of nuclei isolated from Walker carcinoma 256, Hauschka ascites carcinoma TA-3, Ehrlich ascites sar-
Ratios of nuclear proteins to DNA for rat and mouse tumors
115
Figs 1, 2. The lacunae in some of the nuclei do not indicate loss of material from nuclei during the isolation procedure, but rather result from difficulties with fixation and probably embedding. The electron microscope photos of the nuclei have been included mainly to show loss of the outer nuclear membrane. No attempt is intended to show nuclear fine structure. Fig. 1. (a) Nuclei isolated from Walker carcinoma 256. Phase contrast, ca x 750. (b) Nuclei isolated from Hauschka ascites carcinoma TA-3. Phase contrast,, ca x 750. (c) Nuclei isolated from Ehrlich-Lettre ascites sarcoma. Phase contrast, ca x 750. (d) Nuclei isolated from Morris hepatoma. Phase contrast, ca x 750. Exptl
Cell Res 67
116 J. Magliozzi et al. Table 3. Analyses of nuclei isolatedfrom Morris hepatomafor ratios of protein fractions to DNA Percent DNA and ratios Percent DNA Glob/DNA H,/DNA P,/DNA P,/DNA
Prep. la 13.10 0.25 2.00 1.43 3.68
Prep. 2b 10.60 0.33 1.85 1.95 4.12
Ave. 11.90 0.29 1.93 1.69 3.85
Glob, globulin; Ht, total histone; P,, residual protein; P,, total protein. a 0.1 N HCI used to extract histones. b 0.2 N HCl used to extract histones.
Fig. 2. Electron micrographs of nuclei from: (a) Walker carcinoma 256; (b) Hauschka ascites carcinoma TA,; (c) Ehrlich-Lettre ascites sarcoma. Fixation: (a, b) Karnovsky fixative, pH 7.22; (c) 2 % glutaraldehyde0.001 M MgCl,. Staining: (a, b, c) uranyl acetate, lead citrate. Magnification (a) x 3 400; (b) x 1 800; (c) xl 300.
coma, and Morris hepatoma are shown in fig. la-d respectively. These photographs refer to small nuclear aliquots withdrawn from preparations and fixed immediately after they were finished. Some of the nuclei still retain tabs or thin shells of Exptl
Cell Rrs 67
cytoplasm which have lost visible particulate matter and therefore probably the dissolved part of the cytoplasm at least. In one or two casescleaner nuclei than are shown were obtained from the Morris hepatoma when the cells were harvested between 1 and 1.5 weeks after inoculation. Electron micrographs of nuclei from Walker carcinoma 256, Hauschka ascitescarcinoma TA, and Ehrlich-LettrC ascites carcinoma, respectively are shown in fig. 2a- c. It can be seen that the outer nuclear membrane has been lost, presumably because of the use of the Triton N-101 in the isolation procedure. A similar observation has been recorded by Zalta et al. [15]. The same thing has been observed subsequently by Berkowitz et al. [3]. Loss of the outer nuclear membrane and the lipid associated with the inner membrane might account for the observed failure of the nuclear pellet to blacken markedly in buffered osmic acid. An electron micrograph of a tabbed Walker carcinoma nucleus is shown in fig. 3. A mass of fiber can be seen in the tab, part of which (the ropelike structure) seemsto be attached to the nuclear membrane. Most of the tabbed Walker tumor nuclei examined that had been fixed in Karnovsky fixative
Ratios of nuclear proteins to DNA for rat and mouse tumors
117
Fig. 3. Electron micrograph of tabbed Walker carcinoma nucleus (see Methods section). Fixation: Karnovsky fixative, pH 7.22. Staining: Uranyl acetate, lead citrate. Magnification ca x 53 500.
showed such fibers. Dr Keith Porter, who examined some of our early electron micrographs, stated that in his opinion the fibers
represented distorted microtubules from the centrosphere, a region around the centriole. (See [l, 7, 221 for a description of microExptl
Cell Res 67
118
J. Magliozzi et al.
Fig. 4. Part of whole cell from Walker tumor showing microfibers. Silver section. Fixation: 10 % acrolein in pH 7.3 phosphate buffer. Post fixation: Palade 1 % osmic acid for 1.5 h. Araldite embedding. Staining: Uranyl acetate, lead citrate. Magnification x 69 000. We are indebted to Mr J. van Blerkom of the University of Colorado who took this electron micrograph using a Philips 200 instrument.
Exptl
Cell
Res
67
Ratios of nuclear proteins to DNA for rat and mouse tumors Table 4. Analyses for ratios of protein fractions to DNA of nuclei isolated from normal rat liver by the lead-Triton method developed for isolating cancer cell nuclei Percent DNA and ratios
Prep. 1
Prep. 2
Ave.
Per cent DNA Glob/DNA HJDNA P,/DNA PJDNA
8.50 0.17 2.64 2.48 5.28
9.40 0.20 1.78 2.66 4.61
8.95 0.19 2.16 2.57 4.95
Glob, globulin; H,, total histone; P,, residual protein; P,, total protein.
tubules). Ehrlich ascites sarcoma nuclei bearing tabs, or cells surrounded by shells of cytoplasm (cell remnants) showed similar fibers and at times endoplasmic reticulum, mitochondria, and inclusion bodies. The same type of fibers was seen in whole cells of the Walker tumor (seefig. 4). Apparently similar fibers have been reported by Sykes et al. [23]. The Ehrlich sarcoma cell remnants used in this work were obtained from Ehrlich ascites sarcoma cells that had been allowed to remain in the host mice for 2 weeks before harvesting (seeExperimental section). The method described in this paper for isolating the malignant nuclei does not yield nuclei capable of forming gels [19], indicating that there has been some kind of partial autolysis of the macromolecules of the chromatin during the isolation procedure. We believe on the basis of preliminary evidence that this attack was made by DNAase; the lead used in the isolation acts as a protease inhibitor. Whatever autolysis occurs apparently does not, however, appreciably change the ratios of the principal protein fractions to DNA, since a control experiment showed that normal rat liver nuclei isolated by the same method as was used for isolating the tumor nuclei still show the usual ratios for
119
the various protein fractions to DNA that are characteristic of rat liver nuclei that are capable of gel formation (see table 4). The rat liver nuclei isolated by the method for isolating tumor nuclei, like the latter, do not form gels. It has been found possible to obtain liver cell nuclei capable of forming gels by use of the same method as described for the isolation of the cancer cell nuclei, except for the addition of 0.0002 M indium trichloride in the homogenization and 0.0001 M indium trichloride in the subsequentsteps, where the concentration of Pb2+ was reduced to 0.0001 M. However we have not yet found concentrations of indium and lead that will allow good cell breakage in working with cancer cell nuclei. Analyses of the isolated nuclei for the ratios of the principal protein fractions to DNA The ratios of the “globulin” fraction, the total histone, and the residual protein to DNA for the four tumors studied are shown in tables 1 through 3. As a control the same kind of data is given in table 4 for normal rat liver nuclei isolated by the same method that was used for the cancer cell nuclei. It can be seen that the ratio of total histone to DNA in all casesis considerably above 1, and is close to 2 in the case of the Ehrlich ascites tumor and the Morris hepatoma. The ratio of residual protein to DNA is often in the neighborhood of 3 but is occasionally higher; in the case of the Morris hepatoma, it is slightly under 2, in spite of the fact that owing to the presence of some fiber in these nuclei, one might have expected this ratio to be high rather than low. It is of interest to compare these ratios with corresponding ratios for liver cell nuclei [lo]. If Pb2+ is omitted from the preparation nuclei can be isolated from the Walker & Exptl
Cell Res 67
120 J. Magliozzi et al. Hauschka TA3 carcinomas, are greatly reduced and the histone to DNA are lowered than 1.0, as is shown by the 1 and 2.
but the yields ratios of total to values less data in tables
DISCUSSION For a number of years, it was generally held that the problem of breaking cells prior to isolating nuclei could be solved by applying mechanical forces to cells. The investigations of Busch on the relationship between homogenizer pestle clearance and yield of nuclei showed that in the case of the Walker carcinoma, the optimal clearance was about 77 P 151. It is possible to employ non-ionic detergents to aid in the breakage of the tumor cell. Zalta et al. [15] appears to have been the first to use such detergents in isolating cell nuclei. When used at the pH range of 6.57.0, these detergents allow one to break the Walker cells with high efficiency, using the loose pestle. Examples of the detergents suitable for this purpose are Triton X-100 and Triton N-101. Hymer & Kuff [16] introduced the former product in a method suitable for the preparation of rat liver nuclei and mouse plasma cell nuclei; Lazarus & Sporn employed the latter in a method designed to isolate nuclei from the mouse Ehrlich-LettrC sarcoma [ 181. Triton N-101 has recently been used in the isolation of HeLa cell nuclei [3, 201, and non-ionic detergent has come to be accepted quite generally as an aid in isolating tumor cell nuclei. A mixture of ionic and non-ionic detergent has also been used [13]. Either of the above-mentioned detergents causes a sufficient attack on the cytoplasmic matrix so that it can be removed from nuclei by homogenization. A disadvantage in the use of the Tritons is that they appear to destroy mitochondria and lysosomes during Exptl
Cd
Res 67
homogenization, a process which releases enzymes which attack macromolecules of chromatin and thus render tumor nuclei non-gelable. In this respect, isolation of nuclei in the presence of non-ionic detergent is similar to isolation using water as homogenizing medium. Also, the outer nuclear membrane is removed by the Tritons [3, 161. In spite of these disadvantages, the use of detergent is the only means available at present for obtaining satisfactory removal of cytoplasm from most types of cancer cell nuclei. Although enough cancer cells can be broken in the pH range of 6.5 to 7.0 to liberate most of the nuclei, lowering the pH to 5.8 greatly increases the resistance of the cells to breakage so that satisfactory isolation of nuclei cannot be accomplished, even when detergents are present. Since the use of detergent favors the release of enzymes which tend to attack or destroy nuclei, the introduction of some agent which can suppress these enzymes in order to stabilize the nuclei and prevent loss of nuclear macromolecules is highly desirable and in some cases at least probably necessary if nuclear isolation is to be achieved. In the past, Ca2+ and Mg2+ have often been used for stabilization; these cations probably are effective because of causing condensation of chromatin. In the absence of other enzyme inhibitors however, Ca2+ and Mg2+ cause a loss of the easily extractable histone fraction from liver cell nuclei [lo]. They also tend to enhance the resistance of the cells to breakage. Our recent discovery that Pb2+, Cd2+ and In2+ aid in stabilizing nuclei in cellular homogenates [ll], presumably by inhibiting the action of proteases and other enzymes, has induced us to investigate the use of these metal cations in isolating Walker tumor nuclei. It has been found that Pb2+ can be used in the homogenizing medium in the pH range of 6.5-7.0; the optimal concentra-
Ratios of nuclear proteins to DNA for rat and mouse tumors
tion is 0.0002 M. Pb2+ appears to be the best of the three cations just mentioned in the case of tumor cell nuclei; the use of Cd2+ at 0.0002 M concentration permits liberation of the nuclei but leaves many nuclei with attached cytoplasmic tabs. Indium trichloride is effective as a stabilizer but increases the difficulty of liberating the nuclei. Pb2+ has therefore been used for the most part in this study. It has been found that the use of MgCl, in washing the cancer cell nuclei which have been partially isolated in the presence of Pb2+ is very helpful and probably necessary. Its use throws the nuclei into a more condensed state and enables one to use the tightly-fitting pestle of homogenizer, which is essential for removing residual cytoplasmic tabs from the nuclei. The use of Mg2+ in washing the nuclei also reduces cytoplasmic agglutination, which is increased in washing the nuclei in the presence of lead. It has been determined that the use of Mg 2+ in washing liver cell nuclei subsequent to the use of Pb2+ does not cause loss of the easily extractable histone fraction or otherwise affect the ratios of the principal protein fractions to DNA and therefore its use in the isolation of tumor nuclei subsequent to the use of Pb2+ appears justified. Since the total histone to DNA ratio of about 1.6 (see table 1) found in this study for Walker tumor nuclei lies between the ratio of about 1.Ofound thus far for calf thymus nuclei and the ratio of 2-2.5 found for rat liver nuclei [I 1, 121, it might be thought that sufficient stromal nuclei having an HJDNA ratio of 2.5 were present along with carcinoma nuclei having a ratio of 1.0 to account for the observed intermediate ratio of the nuclear preparation. However this interpretation would require the presence of at least 40 y, stromal cells; microscopic observation indicates that the percentage of stromal cells in
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the preparation must be far less than this, and hence we ascribe the observed ratio of 1.6 to the tumor cell nuclei themselves. The intermediate ratio for the Hauschka ascites carcinoma nuclei apparently must also be ascribable to the malignant cell nuclei, since no stromal cell nuclei should be present in the nuclei isolated from this malignancy. The lower ratios reported previously for tumor cell nuclei [25] were undoubtedly due to the use of Mg2+ without heavy metal in the isolation procedure. Disc gel electrophoresis of the total histone fractions from the cancer cell nuclei has indicated that probably not more than lo15 % of the fractions are non-histone protein. The use of 0.2 N HCl instead of 0.1 N HCl for extracting the total histone of the tumor nuclei did not on the average result in more than a very small increase in the ratio of total histone to DNA. The difficulty in removing tabs of cytoplasm from cancer cell nuclei is the rule rather than the exception [8]. We know of no explanation for this difficulty that has been offered previously. The ability of the attached tabs to keep their shape in spite of breakage of the cell membrane leads to the deduction that the tabs must possess some sort of framework structure that maintains their shape and keeps them bound to the nuclei. Such a framework structure must be at least in part constructed of protein, since the tab structure was found to be destroyed by pepsin [8]. The presence of fibers in malignant cells has been noted by Sykes et al. [23], who credit Haguenau [14] as being the first to report them. The microfibrils of Haguenau were found in myoepithelial cells and do not appear to be related to the microfibrils discussed by us. The term “tonofibrils” was used in designating these microfibrils which were thought to be characteristic of malignancy. Exptl
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122 J. Magliozzi et al. The fibers found by us in nuclear tabs and cytoplasm of the Walker tumor and Ehrlich ascites sarcoma cells appear similar to those found by Sykes et al. as far as can be judged by the electron micrographs. It seems reasonable to suppose that the fibers found in the tabs and cytoplasm of the Walker tumor and the Ehrlich ascites sarcoma constitute the main part of the framework structure just discussed. Apparently removal of the outer nuclear membrane at least is generally necessary if clean tumor nuclei are to be isolated. This leads one to suspect attachment of the fibers to the outer nuclear membrane. The latter, as well as most of the inner membrane, can be removed by strong citric acid [4] as well as by the use of detergents, and the use of either of these reagents can lead to the isolation of relatively clean nuclei. Another factor that possibly could contribute to the difficulty of isolating nuclei from malignant cells from tissue culture is the presence of intercellular bridges [2]. Busch [6] has reported a method for the isolation of clean nuclei from certain Morris hepatomas which does not involve the use of non-ionic detergent, but this method did not work with all Morris hepatomas. In the same article Busch has criticized the use of osmotic
swelling
followed
by homogeniza-
tion in the presence of detergents in isolating tumor nuclei, on the basis of a report by Penman [21] that nuclei isolated in this way lose completely their 18S RNA. We have not been concerned with nuclear RNA in this work, and moreover we have avoided the use of osmotic swelling and have added a stabilizing agent (Pb2+) that has been shown to protect cell nuclei against proteolytic action. A final point worth emphasizing is the variability in tumors which affects the ease of isolating nuclei in an important manner. If Exptl
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the tumors grow very slowly and therefore are left in the host animals for some time before being harvested, the isolation of nuclei may be difficult or impossible. This has been noted in the case of the Walker tumor, the Ehrlich ascites tumor, and probably the Morris hepatoma. The situation is particularly interesting in the case of the Ehrlich ascites sarcoma; it is impossible to isolate nuclei by the method described in this paper if the cancer cells are harvested 2 weeks after inoculation, whereas fairly good nuclei can be obtained if the cancer cells are harvested one week after inoculation. It is thus of great importance to make every effort to arrange conditions so as to obtain as suitable tumor or cancer cells as possible if cell nuclei are to be isolated. J. M. gratefully acknowledges support of USPHS Training Grant AM 0100404. R. 0. is indebted to USPHS (5Tl-HD-00133-03) for support of his part in this research. A. L. D. is glad to acknowledge support from the USPHS, National Cancer Institute, grant CA 00994-19.
D. P. is glad to acknowledge support of National Cancer Institute grant CA-1 19801.
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Ratios of nuclear proteins to DNA for rat and mouse tumors 13. Ginzburg-Teitz, Y, Kaufmann, E & Traub, A, Biochim biophys acta 134 (1967) 211. 14. Haguenau, F, Compt rend acad sci 249 (1959) 182. 15. Hubert, M T, Farard, P, Carasso, N, Rozencwajg, R & Zalta, J P, J microscop 1 (1962) 435. 16. Hymer, W- C & Kuff, E L,-J histochem cytochem 12 (1964) 359. 17. Karnovskv. M J. J cell bio127 (1965) 137n. 18. Lazarus, H M & Spom, M B, Proc natl acad sci US 57 (1967) 1386. 19. Mackay, M, Hilgartner, C A & Dounce, A L, Exptl cell res 49 (1968) 533. 20. Munro, G F, Dounce, A L & Lerman, S, Cancer res 55 (1969) 46. 21. Vesco, C & Penman, S, Biochim biophys acta 169 (1968) 188.
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22. Porter, K R, Principles of biomolecular organisation. Ciba foundation symposium (ed G E W Wolstenholme & N O’Connor) p. 308. Little, Brown & Co, Boston (1966). 23. Sykes, J A, Recher, L, Jernstrom, P H & Whitescarver, J, J natl cancer inst 40 (1968) 195. 24. Umafia, R & Dounce, A L, Exptl cell res 35 (1964) 277. 25. Umafia, R, Updike, S, Randall, J & Dounce, A L, The nucleohistones (ed J Bonner & P Ts’o) p. 200. Holden-Day, San Francisco (1964).
Received December 17 1970 Revised version received March 2, 1971
ExptI Cell Res 67