[CANCER RESEARCH 43, 2790-2795, June 1983]0008-5472/83/0043-0000$02.00

Rapid Method for Preparation of Nuclei from a Mucin-producing SolidTumor1

Bill F. Hefley, Jr.,2 David M. Duhl,3 Robert C. Briggs, Loren H. Hoffman, and Lubomir S. Hnilica

Departments of Biochemistry and Anatomy [L. H. H.] and the A. B. Hancock, Jr., Memorial Laboratory of the Vanderbilt University Cancer Center [D. M. D., R. C. B.,L S. H.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232

ABSTRACT

Nuclei were prepared from the human colon mucin-producingtumor GW-39. The method described involves incubation in ß-

mercaptoethanol under mild conditions and results in the preparation of nuclei free of the mucin web that initially enmeshesthem. A sensitive colorimetrie immunoassay was used to confirmthat nuclei prepared in this fashion were generally free of contaminating mucin. This method is mild, rapid, and should be applicable to other mucin-producing tumors.

INTRODUCTION

The preparation of nuclei from mammalian cells is most commonly based on the principle of differential centrifugation inbuffered hyptertonic and isotonic solutions (12, 23). When thisapproach is applied to tumors, either freshly excised or propagated in laboratory animals, the situation is confounded by theheterogeneity of cell types (2, 7, 23), the presence of necroticcells, and in some cases production of mucin (30). The productionof mucin is particularly troublesome because it not only is apotential nuclear contaminant (20) but also, as a result of itsgelatinous properties, makes nuclear separation and purificationbased on differential centrifugation very difficult. The viscosity,and hence potential contamination, of mucin can be decreasedby the application of A/-acetylcysteine (19) or by digestion with0-glucuronidase or neuraminidase (30). More drastic conditions,

such as the use of proteolytic agents (1, 24) or the phenol:waterextraction of mucin (14), are generally considered too harsh tomaintain the morphological integrity of nuclei and chromatin.

The present paper describes a mild treatment with ß-mercap-

toethanol to disrupt the mucin gel and allow for the preparationof purified nuclei by traditional means. A sensitive peroxi-

dase:antiperoxidase assay has been adapted to demonstratethat the nuclear preparation is generally free of contaminatingmucin, known to be produced in large amounts by this tumor(16, 30). This method should be applicable to other mucin-

producing tumors, is milder than enzymatic digestions, andserves as a rapid and convenient method of preparing nucleifrom these tumors.

MATERIALS AND METHODS

Propagation of Tumor. The mucin-producing human colon adenocar-cinoma cell line GW-39 was a gift from Dr. David Goldenberg (University

1Supported by National Cancer Institute Grant CA 27338 and USPHS Training

Grant CA 09313.2 Recipient of a Clinical Cancer Education Program summer assistantship

through National Cancer Institute Grant R25-CA 19429. A medical student atVanderbilt University School of Medicine.

3To whom requests for reprints should be addressed, at the Department of

Biochemistry, Vanderbilt University School of Medicine, Nashville, Tenn. 37232.Received November 1, 1982; accepted March 10, 1983.

of Kentucky Medical Center, Lexington, Ky.) and was propagated as aserially transplanted solid tumor in the cheek pouch of unconditionedSyrian hamsters. Tumors were grown in the hind leg muscle for harvesting. Growth in cheek pouches or leg muscle was from a fine minceate oftumor cells in sterile Hanks' balanced salt solution (Grand Island Biolog

ical Co., Grand Island, N.Y.) containing penicillin and streptomycin.Preparation of Nuclei. All solutions used for the preparation of nuclei

contained 0.1 mM phenylmethylsulfonyl fluoride and were used at 0-4°.

All homogenizations were done with a loose-fitting Teflon pestle unless

stated otherwise. Connective tissue and muscle were trimmed, andexcess blood was removed by rinsing the tumor in 0.25 M sucrose:0.01M Tris, pH 7.5. The tumor was minced finely and homogenized in 0.2 M/i-mercaptoethanol:5 mw MgCI2:0.15 M NaCI:0.1 M Tris, pH 8.5, using a

tightly fitting Teflon pestle to reduce mucin viscosity (1). The homogenatewas stirred for 30 min on ice and then centrifugea at 3000 x g for 10min. The pellet (Pellet 1) was homogenized in 0.1 M /3-mercaptoethanol:5

mw MgCI2:0.25 M sucrose:0.01 M Tris, pH 8.5, and filtered through 4layers of gauze. The suspension was centrifuged at 900 x g for 10 min,and the resulting pellet (Pellet 2) was resuspended in 0.25 M sucrose:0.01M Tris, pH 7.5. The preparation of nuclei and chromatin from this materialwas as described by Spelsberg ef al. (25). Briefly, the crude nuclei werecollected by centrifugation at 900 x g for 10 min, and the resulting pellet(Pellet 3) was homogenized in 2.2 M sucrose:5 mivi MgCIjiO.OI M Tris,pH 7.5. The homogenate was underlaid with additional 2.2 M sucrose:5mm MgCI2:0.01 M Tris, pH 7.5, and centrifuged at 93,000 x g for 1 hr.Nuclei (Pellet 4) were suspended in 0.25 M sucrose:0.5% (v/v) Triton X-

100:0.01 M Tris, pH 7.5, and collected as a pellet (Pellet 5) aftercentrifugation at 900 x g for 10 min. A summary of the nuclear preparation may be found in Chart 1.

The material in Pellet 5 was fixed at 4°in 2.5% glutaraldehyde in 0.1

M sodium cacodylate buffer (pH 7.3). After 2 hr, the pellets were rinsedextensively in the buffer and postfixed 90 min in the same buffercontaining 1.0% OsO4. Following ethanol dehydration, samples weretransferred through propylene oxide prior to embedding in Epon.

Areas for electron microscopic analysis were selected randomlythroughout the pellets after light microscopic examination of 1->im-thick

sections stained with toluidine blue. Thin sections were mounted onnaked copper grids, stained with uranyl acetate and lead citrate, andexamined in an Hitachi Model H-600 electron microscope operated at 75

kV.Assay for Mucin Contamination. Contamination by mucin was deter

mined colorimetrically using the peroxidase:antiperoxidase method asdescribed (13, 27) with ABTS4 as electron donor (29). Nuclei (5-ml

aliquots at various stages of purification) were collected by centrifugationat 4000 x g for 7 min. Each incubation was for 1 hr at room temperaturewith constant agitation. The order and dilution (in 1 ml of PBS) ofimmunoreagents were as follows: peanut agglutinin (50 ¿ig/ml;SigmaChemical Co., St. Louis, Mo.); 1:40 dilution of antipeanut agglutinin madein rabbit (E-Y Laboratories, San Mateo, Calif.); 1:40 dilution of anti-rabbit

IgG made in goat (Serasource, Inc., Berlin, Mass.); and 1:200 dilution ofperoxidase:antiperoxidase made in rabbit (prepared by J. Briggs in thislaboratory). After each incubation, the pellets were collected by centrifugation at 4000 x g for 7 min and washed 2 times by suspension and

4The abbreviations used are: ABTS, 2,2'-azino-di-(3-ethylbenzthiazolinesulfonic

acid); PBS, phosphate-buffered saline (0.01 M NaH2PO4-H2O:0.15 M NaCI, pH 7.1);CEA, carcinoembryonic antigen.

2790 CANCER RESEARCH VOL. 43

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from

Nuclei Preparation from Tumors

GW-39TUMOR

Trim connective tissue

Rlns« In 0.25 M lucro»: 0.01 H Tris, pH 7.5

Homogenize and Incubate 30 Bin In0.2 M 6-mercaptoethanol: 5 «M HgCl2: 0.15 M NaCl:

0.1 K Tris, pH 8.5

3000 x g, 10 min

Homogenize In 0.1 M B-mercaptoethanol:

5 mM MgClj: 0.25 H sucrose: 0.01 H Tris, pH 8.5

Filter through 4 layers of gauze

Chart 1. Flow diagram for preparation of nuclei fromGW-39 tumor. Details are given in "Materials and Methods."

I 900 x g, 10 «In

PELLET 2I

Homogenize In 0.25 sucrose: 0.01 M Tris, pH 7.5

900 x g, 10 «In

PELLET 3 I

Homogenize In 2.2 M sucrose: 5 mH MgCl: 0.01 H Tria, pH 7.5

91,000 X g, 1 h

Homogenize In 0.25 M sucrose: 0.5Z (v/v) Triton X-100:

0.01 M Tris, pH 7.5

900 x g, 10 min

[ PELLET 5 I~~ Homogenize In 0.25 M sucrose: 0.01 M_ Tris, pH 7.5

NUCLEARPREPARATION

SUPERNATANTDiscarded

SUPERNATANTDiscarded

SUPERNATANTDiscarded

SUPERNATANTDiscarded

SUPERNATANTDiscarded

centrifugation in 4 ml of PBS (5 washes in PBS after incubation withperoxidaseiantiperoxidase). The final color reaction was produced byadding ABTS (1.14 mg/ml; Sigma) and 0.005% H2O2 in sodium phosphate buffer {3.33 mw NaH2PO4:0.67 IHM Na2HPO4, pH 6.0). The colorreaction was stopped by adding 0.1 volume of 5 mw sodium azide (22),the suspension was clarified by centrifugation at 4000 x g for 10 min,and the absorbance (at 420 nm) of the supernatants was determined.

Analytical Methods. DMA and RNA content was determined on HCIO4precipitates by the diphenylamine and orcinol reactions, respectively, asdescribed by Ch'ih ef al. (9). Protein content was determined by the

method of Lowry et al. (21) using bovine serum albumin as standard.Gel electrophoresis was as described previously (10), and molecularweights were approximated by comparing the relative mobilities ofproteins to those of Known molecular weights (Bio-Rad Laboratories,

Richmond, Calif.). Complement fixation was according to the method ofWasserman and Levine (28) as described previously (10) using 75 pgtotal protein and anti-CEA (raised in goat) at a dilution of 1:100.

RESULTS

The production of large amounts of mucin by some adenocar-cinomas requires the dispersion of cells and nuclei before chro-matin can be prepared by classical means. In the case of GW-

39 tumor, this was accomplished by incubation in the presenceof /3-mercaptoethanol as shown in Chart 1. Phase contrast

photomicrographs of the resulting nuclear preparation are shownin Fig. 1. When the tumor minceate was homogenized andincubated in 0.2 M /î-mercaptoethanol:5 rriM MgCI2:0.15 M

NaCI:0.1 M Tris, pH 8.5, the resulting homogenate consisted ofintact cells arranged in clusters which were enmeshed in mucinlacunae as described by Yeoman ef al. (30). This gelatinoussuspension also contained large amounts of debris includingsome highly refractile material of unknown origin. When theresulting pellet (Pellet 1) was suspended in 0.1 M /3-mercaptoeth-

anol:5 mM MgCI2:0.25 M sucrose:0.01 MTris, pH 8.5, and filtered,the suspension was much less viscous and contained primarilyindividual cells; groups of cells enmeshed in mucin lacunae and

free nuclei were rarely present. Pellet 2, obtained after centrifugation of the filtrate, was firmer and less gelatinous than Pellet1 and, when suspended in 0.25 M sucrose:0.01 M Tris, pH 7.5,consisted of intact nuclei with few whole cells present. Cyto-

plasmic and noncellular debris were abundant as was the highlyrefractile material. When the crude nuclei were collected bycentrifugation and suspended in 2.2 M sucrose:5 HIMMgCI2:0.01M Tris, pH 7.5 (Pellet 3), the nuclei became pyknotic as a resultof the hypertonic conditions. The suspension also containedextranuclear debris as well as some of the highly refractilematerial. The supernatant, after the 93,000 x g centrifugation,contained much paniculate debris, highly refractile material, distorted and damaged nuclei, and nuclei with prominent cytc-

plasmic tags (data not shown). The corresponding pellet (Pellet4) in 0.25 M sucrose:0.5% (v/v) Triton X-100:0.01 M Tris, pH 7.5,contained intact, well-rounded nuclei which appeared translucent

and generally free of surface debris. Very little particulate debriswas present, and the highly refractile material was virtuallyabsent. When this material was pelleted by centrifugation andresuspended in 0.25 M sucrose:0.01 M Tris, pH 7.5 (Pellet 5), theresulting nuclei appeared translucent and were fragile with somereleasing their chromatin content. Examination by electron microscopy showed that the final Triton X-100 nuclear pellet was

composed of nuclei devoid of both the nuclear envelope andextranuclear material including membrane fragments (Fig. 2).

Contamination by mucin was assessed by taking advantageof the specific binding of peanut agglutinin to colon tumor mucin(5, 6). The binding was detected by subsequent binding of anti-

peanut agglutinin to the lectin and quantitated colorimetrically at420 nm using the peroxidase:antiperoxidase method (10) withthe soluble reagent ABTS. Chart 2 shows that the highestreactivity was found at the beginning of the nuclear preparation,and treatment of the sample with the reducing agent /3-mercap-

toethanol reduces the mucin content by 60%. Nuclei, afterpassage through 2.2 M sucrose-containing buffer (Pellet 4), show

JUNE 1983 2791

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from

8. F. Hefley, Jr., et al.

g i.oo*2 LU0.80

y 0.60<n §LU 5

o. g 0.40

p 0.20

§K

WH P4PI P2

STAGE OF PREPARATION

Chart 2. Presence of mucin in nuclear preparation at various states of purification. Values are expressed as percentage of A42o-absorbing material found in wholehomogenate, and each fraction was prepared as described in Chart 1. WH, wholehomogenate; Pi. Pellet 1; P2, Pellet 2; P3, Pellet 3; and P4, Pellet 4.

Table 1

Analysis of whole homogenate and nuclear fractionsExperimental details are given in "Materials and Methods."

Whole homogenateNucleiDNA"

(mg/g

tumor)ND8

0.21 ±0.09RNA"

(mg/g

tumor)0.13 ±0.04'

0.02 ±0.01Protein0

(mg/

gtumor)84.6

±9.35.4 ±1.3CEA"77.66.4

3 Four experiments.0 Four experiments.c Three experiments." Percentage of complement fixed using anti-CEA." ND, not determined.

Mean ±S.D.

only 6.6% of the original /Wo-absorbing material present in the

whole homogenate.The nuclear fraction was analyzed for DNA, RNA, protein

content, and extranuclear contamination (Table 1). The DNAcontent in the whole homogenate fraction was not determinedbecause this fraction contained sugars (glycoproteins, glycoca-lyx) that interfere with the diphenylamine test resulting in ametachromatic reaction product that artifactually elevated theabsorbance.5 The DNA:protein ratio in the nuclear fraction (0.04)

was typical of values reported for other tissues, such as rat liver(9). The DNA: RNA ratio and the negligible amount of RNA foundin the nuclear preparation (0.004 mg/mg of protein) demonstrated that this nuclear isolation procedure resulted in onlynegligible cytoplasmic contamination. This was confirmed whencontamination with an extranuclear protein, CEA, was examinedby complement fixation (Table 1). While 77% of the complementwas fixed in the whole homogenate fraction under these conditions, virtually no complement was fixed (and hence no CEA waspresent) in the nuclear fraction. The 6% complement fixed couldbe considered background, since antibody alone (no proteinadded) gave a similar value (data not shown).

When chromatin was prepared from isolated nuclei (10), adramatic difference was observed between nuclei prepared withand without /3-mercaptoethanol (Fig. 3). Major contaminating

proteins of approximate molecular weights of 174,000 and38,000 were characteristic of chromatin prepared by existingmethods (10, 25) and greatly reduced or absent when preparedusing /3-mercaptoethanol as described in "Materials and Methods."

DISCUSSION

The preparation of nuclei from the GW-39 tumor requires prior

release of cells from the mucin web that enmeshes them. Procedures for preparation of nuclei outlined by Spelsberg ef al.(25), successful for other cell types including colon adenocarci-

noma cells grown in vitro or nonneoplastic colonie epithelial cells(10), proved to be unsuitable for the GW-39 tumor. Factors

contributing to this lack of success included the viscosity andthe gelatinous quality of the mucin and the enclosure of cells inthe mucin sacs and lacunae characteristic of this tumor (30). Asa result, the method of Spelsberg ef al. (25) had to be modifiedto bring about release of the cells from the mucin network whileminimizing mucin contamination.

Mucin is known to form large aggregates held in a 3-dimen-

sional network by disulfide bonds (11, 24). These bonds involvenot only the mucin polypeptide backbone but also contaminatingproteins which are known to be entangled in the 3-dimensional

mucin network (24). The viscosity of mucin can be decreased byreduction of these bonds with dithiothreitol (11) or 0.2 M ß-

mercaptoethanol (1, 24). Other work has shown that the presence of cations (1) and partial proteolysis (1, 24) also reduceviscosity. When the presence of a reducing agent was combinedwith 0.15 M NaCI to maintain isotonicity and 5 mw MgCI2 tomaintain nuclear morphology and prevent nuclear clumping (23),the result was a less viscous suspension of individual cells whichcould be separated from the mucin in subsequent steps. Chart1 outlines the procedure used. The 30-min incubation to reduce

the disulfide bonds was followed by a second exposure to thereducing agent in isotonic sucrose which kept the mucin reducedwhile cells were pelleting through isotonic sucrose. This methodis not only rapid but can be conducted at 0-4° which retards

proteolytic activity. Yeoman ef al. (30) have described a successful mucin digestion procedure involving neuraminidase andß-glucuronidase, but the conditions involve a 3-hr incubation at37°which may have adverse effects on heat lability or phenyl-

methylsulfonyl fluoride-resistant proteolysis of proteins.

After the viscosity had been reduced and the cells releasedfrom the mucin network, more conventional methods for nuclearpreparation could be used (25). In short, after the nuclei werewashed in isotonic sucrose buffer to remove the excess ß-mercaptoethanol, they were purified by centrifugation in hyper-tonic sucrose (8, 23). The high density of 2.2 M sucrose-containing buffer allows nuclei to pellet while keeping nonnuclear, damaged nuclear, and contaminated nuclear material in the supernatant. High viscosity also helps to remove the outer nuclearmembrane by forces of shearing (4). Finally, to remove completely the outer nuclear membrane and attached cytoplasmiccontaminants, the nuclei were washed in isotonic sucrose buffercontaining 0.5% (v/v) Triton X-100 (4, 23). Electron microscopy

confirmed that the nuclear preparation at this step was indeedlacking the outer nuclear membrane and extranuclear contaminants. The final yield of nuclear DNA was consistent with theamount reported to be present in other human tissues (3), andthis value did not decrease when the material was further processed to purified chromatin by the method of Spelsberg ef a/.(25),6 suggesting that this value does indeed represent chromatin

(or material that is inseparable from chromatin).The GW-39 tumor, isolated and characterized by Goldenberg

ef al. (16-18), is an expiant from a primary colonie adenocarci-

noma and has been propagated in immunocompetent hamsterssince 1966. The karyotype confirms it is of human origin (17).

s D. M. Duhl and L. S. Hnilica. unpublished observations. 6 D. M. Duhl, B. F. Hefley, and L. S. Hnilica, unpublished observations.

2792 CANCER RESEARCH VOL. 43

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from

Nuclei Preparation from Tumors

The tumor is known to produce large amounts of mucin, reflecting the retention of some characteristics of differentiated colonieepithelial cells (16). The mucin tends to keep the cells in clusters,which in turn are arranged in lacunae (30). Such a cluster of cellsis shown in the phase contrast photomicrograph of Fig. 1>4andwas obtained by homogenization of the tumor minceate. Background debris of various origins and composed of extracellularmaterial and some cell lysate was abundant and was not removed until the nuclei were centrifuged through 2.2 M sucrose-containing buffer (Fig. 1£).The presence of Mg2* in buffers prior

to the Triton X-100 kept the nuclei intact while still allowing thecells to lyse (Fig. 1C). The highly refractile material (Fig. 1, A toD) probably represented nuclei from necrotic cells which wereosmotically inactive and cosedimented with nondamaged nucleiuntil the 2 were separated in 2.2 M sucrose-containing buffer.Underlaying with fresh 2.2 M sucrose-containing buffer was

essential for this separation, and it was very rare that the nuclearpellet contained this refractile material after this step (Fig. 1, Eand F). In fact, electron microscopy revealed that the nuclearpreparation was free of cosedimenting membranous material(Fig. 2). In summary, from the phase contrast photomicrographsof Fig. 1, it is clear that the mucin network was disrupted in theinitial steps, allowing for the subsequent collection of nuclei inlater steps.

The mucin content was indeed greatly reduced as the nucleiwere increasingly purified (Chart 2). These data are based onthe specific binding of peanut agglutinin to the galactosyl/31-»3A/-acetylgalactosamine domain of colonie tumor mucin. Thisreaction is specific for colon tumor mucin (5, 6), and this determinant has received recent attention as a colon tumor-specific

cell surface antigen (26). The final color reaction accompanyingthe reduction of ABTS is probably only semiquantitative in thisassay, and hence, the trend toward decreased mucin content,rather than absolute values, is of primary importance. Comparedto the whole homogenate, the mucin content was reduced by59% in Pellet 1 after treatment with /3-mercaptoethanol. Further

treatment with this reducing agent decreased the amount ofmucin in the nuclear preparation by an additional 10% in Pellet2. The amount of mucin in Pellet 4 was only 6.6% of that foundin the whole homogenate. This 93% reduction in mucin contentindicates that initial incubation in /3-mercaptoethanol results in

subsequent separation of nuclei from mucin.The amount of DMA recovered in the nuclear preparation and

the DNA:RNA ratio (Table 1) are characteristic of other nucleiprepared by other methods (4, 9), demonstrating that nucleiprepared in the presence of /3-mercaptoethanol exhibit no ad

verse effects on yield or recovery (also see Fig. 2). When theextranuclear protein CEA was used as a marker protein (Table1), it was evident that contamination of nuclei was at an absoluteminimum. This was also apparent when chromatin proteins (fromnuclei prepared in the presence or absence of /3-mercaptoetha

nol) were examined by sodium dodecyl sulfate:polyacrylamidegel electrophoresis (Fig. 3). It is interesting to note that the 2contaminating proteins in chromatin from nuclei prepared in theabsence of /3-mercaptoethanol (but not from nuclei prepared inthe presence of /3-mercaptoethanol), with approximate molecularweights of 174,000 and 38,000 (Fig. 3), share the same approximate molecular weights as cell surface glycoproteins of GW-39

tumors (15).The use of /3-mercaptoethanol allows for the preparation of

nuclei from mucin-producing tumors in a straightforward fashion

with the resulting nuclear fraction free of the mucin network andmorphologically intact. This procedure should be applicable toother mucin-producing tumors and should find wide use when

purified nuclei are critical for biochemical or immunological characterization.

ACKNOWLEDGMENTS

The authors would like to thank Dr. David Goldenberg of the University ofKentucky Medical Center, Lexington, Ky., for the generous gift of the GW-39 tumorin Syrian hamsters and Hoffmann-La Roche Inc. for the generous gift of anti-CEA.

Appreciation is also extended to Virginia Winfrey for assistance in the electronmicroscopic and photographic procedures, Judy Briggs for preparation of the rabbitperoxidase:antiperoxidase, and Nettie Kidd and Beverly Bell for their technicalassistance.

REFERENCES

1. Allen, A., Bell, A., Mantle, M., and Pearson, J. P. The structure and physiologyof gastrointestinal mucus. Adv. Exp. Med. Biol., 744:115-133, 1982.

2. Allfrey. V. G., and Boffa, L. C. Modifications of nuclear protein structure andfunction during carcinogenesis. In: H. Busch (ed.), The Cell Nucleus, Vol. 7, p.522-562. New York: Academic Press, Inc., 1979.

3. Altman, P. L., and Dittmer, D. S. In: Biology Data Book, p. 398. Washington,D. C.: Federation of the American Societies for Experimental Biology, 1964.

4. Btobel, G., and Potter, V. R. Nuclei from rat liver: isolation method thatcombines purity with high yield. Science (Wash. D. C.), 754:1662-1665,1966.

5. Boland. C. R.. Montgomery, C. K., and Kim, Y. S. A cancer-associated mucinalteration in benign colonie polyps. Gastroenterokxjy, 82: 664-672,1982.

6. Boland. C. R., Montgomery, C. K., and Kim, Y. S. Alterations in human coloniemucin occurring with cellular differentiation and malignant transformation. Proc.Nati. Acad. Sei. U. S. A., 79: 2051-2055,1982.

7. Briggs, R. C., Chiù, J.-F., Hnilica, L. S., Chytil, F., Rogers, L. W„and Page,D. L. Human granulocyte specific nuclear antigen(s). I. Production of antiseraand determination of specificity. Cell Differ., 7: 313-323,1978.

8. Chauveau. J., Moule, Y., and Rouiller, C. Isolation of pure and unaltered livernuclei morphology and biochemical composition. Expt. Cell Res., 77: 317-

321,1956.9. Ch'in, J. J., Duhl, D. M., Faulkner, L. S., and Devlin, T. M. Regulation of

mammalian protein synthesis in vivo. Stimulated transport of nuclear ribonu-cleoprotein complexes after cycloheximide treatment. Biochem. J., 778: 643-

649, 1979.10. Duhl, D. M., Banjar, Z., Briggs, R. C., Page, D. L., and Hnilica, L. S. Tumor-

associated chromatin antigens of human colon adenocarcinoma cell lines HT-29 and LoVo. Cancer Res., 42. 594-600,1982.

11. Forstner, J. F., Jabbal, l., Qureshi, R., Keils, D. I. C., and Forstner, G. G. Therole of disulfide bonds in human intestinal mucin. Biochem. J., 787: 725-732,

1979.12. Georgiev, P. The nucleus. In: D. B. Roodyn (ed.), Enzyme Cytology, p. 27-95.

New York: Academic Press, Inc., 1967.13. Glass, W. F., Briggs, R. C., and Hnilica, L. S. Use of lectins for detection of

etectrophoretically separated glycoproteins transferred onto nitrocellulosesheets. Anal. Biochem., 775: 219-224,1981.

14. Gold, D. V., and Miller, F. Comparison of human colonie mucoprotein antigenfrom normal and neoplastic mucosa. Cancer Res., 38: 3204-3211,1978.

15. Goldenberg, D. M., Pant, K. D., and Dahlman, H. L. Antigens associated withnormal and malignant gastrointestinal tissues. Cancer Res., 36: 3455-3463,

1976.16. Goldenberg, D. M., Pavia, R. A., Hansen, H. J., and Vandevoorde, J. P.

Synthesis of carcinoembryonic antigen in vitro. Nat. New Biol., 239:189-190,

1972.17. Goldenberg, D. M., Pavia, R. A., and Smith, L. Karyology of a human colonie

carcinoma (GW-39) serially xenografted in hamsters and in nude mice. CancerLett., 2: 195-200,1977.

18. Goldenberg, D. M., Witte, S., and Elster, K. GW-39: a new human tumorserially transplantable in the golden hamster. Transplantation (Baltimore), 4:760-763.1966.

19. Kubica, G. P., Dye, W. E., Conn, M. L., and Middlebrook, G. Sputum digestionand decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture ofmycobacteria. Am. Respir. Dis., 87: 775-779, 1963.

20. LaMont, J. T., and Ventola, A. S. Purification and composition of colonieepithelial mucin. Biochim. Biophys. Acta, 626: 234-243,1980.

21. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem., 793: 265-275,

1951.22. Persijn, J. P., and Jonker, K. M. A terminating reagent for the peroxidase-

labelled enzyme immunoassay. J. Clin. Chem. Clin. Biochem., 76: 531-532,

1978.23. Roodyn, D. B. Some methods for isolating nuclei. In: G. D. Bimie (ed.),

Subcellular Components. Preparation and Fractionation, p. 15-51. Baltimore:

JUNE 1983 2793

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from

S. F. Hefley, Jr., et al.

University Park Press, 1972.24. Silberberg, A., and Meyer, F. A. Structure and function of mucus. Adv. Exp.

Med. Biol., 744. 53-74,1982.25. Spelsberg, T. C., Hnilica, L. S., and Ansevin, A. T. Proteins of chromatin in

template restriction. III. The macromolecules in spécifierestriction of thechromatin DNA. BkJChim. Biophys. Acta, 228: 550-562,1971.

26. Steptewski, Z., Chang, T. H., Hertyn, M., and Koprowski, H. Release ofmonoclonal antibody-defined antigens by human colorectal carcinoma andmelanoma cells. Cancer Res., 41: 2723-2727, 1981.

27. Stemberger, L. A. The unlabeled antibody peroxidase-antiperoxidase (PAP)

method. In: L. A. Stemberger (ed.), Immunochemistry, p. 104-169. EngtewoodCliffs, N. J.: Prentice-Hall, Inc., 1979.

28. Wasserman, E., and Levine, L. Quantitative micro-complement fixation and itsuse in the study of antigenic structures by specific antigen-antibody inhibition.J. Immunol., 87: 290-295,1961.

29. Werner, W., Rey, H.-G., and Wielinger, H. Ãœberdie eigenscnaften eines neuenchromogens fur die blutzuckerbestimmung nach der GOD/POD-methode. Z.Anal. Chem., 252. 224-228,1970.

30. Yeoman, L. C., Taylor, C. W., and Chakrabarty, S. Human colon tumorantigens. Methods Cancer Res., 79: 233-271,1982.

Fig. 1. Phase-contrast photomicrographs of nuclei at various stages of preparation. Nuclei were prepared as described in Chart 1. A, whole homogenate; H, Pellet 1;C, Pellet 2; D, Pellet 3; £,Pellet 4; F, Pellet 5. x 480.

2794 CANCER RESEARCH VOL. 43

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from

Nuclei Preparation from Tumors

%*

-•,'•. .:''•

V -

•X

3

Fig. 2. Electron micrograph of Triton X-100-washed nuclei from GW-39 cells. Nuclei in Pellet 5 were fixed with glutaraldehyde and prepared for electron microscopyas described in "Materials and Methods." Bar, 1.0 urn.

Fig. 3. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of GW-39 chromatins. Lane 1, prepared in the presence of J-mercaptoethanol; Lane 2, prepared inthe absence of ,<-mercaptoethanol; Lane 3, molecular weight standards. Chromatin samples represent 40 ng (as DNA) applied to the gel, and molecular weight standards(Bio-Rad Laboratories, Richmond, Calif.) are: myosin, M, 200,000; |8-galactosidase, M, 116,500; phosphorylase b. M, 94,000; albumin, M, 68,000; and ovalbumin, M,45,000. Experimental details are given in "Materials and Methods."

JUNE 1983 2795

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from

1983;43:2790-2795. Cancer Res   Bill F. Hefley, Jr., David M. Duhl, Robert C. Briggs, et al.   Solid TumorRapid Method for Preparation of Nuclei from a Mucin-producing

  Updated version

  http://cancerres.aacrjournals.org/content/43/6/2790

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/43/6/2790To request permission to re-use all or part of this article, use this link

Research. on January 3, 2019. © 1983 American Association for Cancercancerres.aacrjournals.org Downloaded from