for DNA from a wide variety of normal and neoplasticmouse tissues.

@siATERIALS AND METHODS

a) Selection of palients.—Patient selection and procurement , handling, and preliminary study of tissue samples

have been described previously (1).b) Preparation of DNA .—DNAwas isolated from whole

tissue with modifications (1) of the procedure of either

Zamenhof (34) or Marmur (24), indicated by the letter“z―or “1@sI―,respectively, prefixedto the sample designation. Enzymatic degradation was prevented by the use ofethylenediaminetetraacetate (EDTA) and citrate in theisolation procedure. The white, fibrous DNA precipitates obtained were dissolved in 0.15 M NaCl and 0.015 MNa-citrate buffer (pH 7.0). The solutions were generallykept at 3°—4°C,although in some cases the samples werepreserved for longer periods by freezing them at —20°C.A commercial calf thymus DNA (Worthington Biochemical Corp., Freehold, N. J.) was used as a reference standard. This was prepared in stock solutions of 1 mg/ml in0.15 M NaCl and 0.015 M Na-citrate buffer (pH 7.0) anddiluted to the required concentrations with the same buffer.

The concentration of the DNA solutions was determined from their absorbances at 260 mj@ by comparisonwith the reference standard (20 @tg/ml of the standardDNA in the above buffer gave an absorbance of 0.234).In addition, colorimetric estimation of DNA was undertaken by the diphenylamine method (7), and, in mostcases, determination of phosphorus content (2) was also

In a j)revious study (1) some differences were found inthe “priming―activity in a DNA polymerase system ofDNA from human neoplasias, compared with Ilormal tissue of the same organ in the same patient. In order toelucidate the possible causes of this difference, we exammed some physicochemical characteristics of DNA fromthese sources. Parameters discussed in this report includemolecular weight , radius of gyrat ion, intrinsic viscosity,and helix-coil transition temj)eratures (melting profile andTm values).

The heterogeneity of DNA from animal tissue is generally recognized. Some disagreement exists on possibledifferences in this heterogeneity between normal and

pathologic tissues (3, 4, 19—21). Differences were reported (27, 28) in the sedimentation-coefficient distribution curves between DNA from white cells and spleensfrom normal individuals and patients with lymphatic andgranulocytic leukemias. Differences were also described inthe molecular weight of DNA from white cells from patients with lymphatic leukemia and from those with

granulocytic leukemia. On the other hand, Kit (21) founda remarkably narrow range of sedimentation coefficients

1 A preliminary report was presented at the 55th annual meeting of the American Association for Cancer Research, Chicago,Ill., April, 1964. This study was supported by grants from theNational Cancer Institute, NIH, USPHS (CA-05522, CA-05136),and the American Cancer Society (E-351).

2 National Science Foundation, Science Faculty Fellow.

Received for publication I)ecember 22, 1964; revised April 12,1965.

1244

Biochemical Parameters of NeoplasiaII. Some Macromolecular Properties of Deoxyribonucleic Acid

from Normal and Neoplastic Human Tissue'

R. J. FIEL,2 T. J. BARDOS, Z. F. CHMIELEWICZ, AND J. L. AMBRUSState Univer.s@ityof New York at Buffalo, Schools of Pharmacy and Medicine, and the Roswell Park Memorial Institute, Buffalo, New York

SUMMARY

DNA (deoxyribonucleic acid) was extracted from neopla.stic human tissues obtamed by surgical biopsy and from corresponding normal tissues from the same organin the same patient. Helix-coil transition temperatures [“melting curves― and Tm(midpoint transition temperature) values], when determined from fresh samples, showedho significant difference between normal and neoplastic DNA and revealed that in bothinstances the preparations represented undenatured, double-stranded DNA. Light

scattering and viscosimetric studies indicated that DNA from neoplastic tissues has

a smaller mean molecular weight and a more rigid configuration than DNA from thematching normal tissues. The radii of gyration of all human DNA samples appearto fall between the values calculated for a theoretical coil and rod, respectively. Theeffect of the procedure of isolation on the macromolecular properties of the DNAis demonstrated.

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No.DiagnosisDNA

concentration byultraviolet

absorbance at260 mp(mg/mi)DNA

concentration by

diphenylammetest

(mg/mi)@p

@ (mean ofdeterminations)Ratio

ofabsorbances

A,,,/A,s,

FIEL et al.—Biochemical Parameters of Neoplasia 1245

performed as a safeguard against possible errors in theDNA concentration data based on the ultraviolet absorbance measurements. Such errors could be due to hyperchromicity (denaturation) of the DNA preparations or tosome unusual variations in their base compositions, causing significant differences in the molar absorbances.

Table 1 lists concentration data for the 30 differentDNA preparations from normal and malignant humantissues that were included in the present report. The con

centrations as estimated by the diphenylamine method are

in good agreement with the corresponding values based onabsorbance measurements. The molar absorbances basedon phosphorus, e(P)Ho, do not show significant variationsamong different preparations; these values are within 15 %of the average value of 6600 found in the literature (10)for calf thymus DNA. The last column in Table 1 liststhe ratios of absorbances at X 260/280 m@. This ratio,which is dependent not only on the extent of denaturation(secondary structure) of the DNA but also on its puritywith respect to protein, varies from a low value of 1 .50 to

TABLE 1

CONCENTRATIONS OF DNA SoLUTIONS BY SPECTItOPHOTOMETRIC AND CHEMICAL METHODS

Medium differentiated adenocarcinoma of stomachNormal stomach tissue

Medium differentiated adenocarcinoma of rectumNormal rectal tissue

Medium differentiated adenocarcinoma of rectumNormal rectal tissue

Medium differentiated adenocarcinoma of rectumNormal rectal tissue

Medium differentiated adenocarcinoma of lungNormal lung tissue

Neuroblastoma of adrenal, medium differentiatedNormal adrenal

Poorly differentiated squamous cell carcinoma ofmaxillary sinus

Spleen, Hodgkin's disease, and hypersplenism

Anaplastic carcinoma of stomachNormal stomach tissue

Adenocarcinoma of colonNormal colon tissueNormal colon mucosa

Undifferentiated carcinoma of cecumNormal cecal mucosa

Medium differentiated adenocarcinoma of colon

Adenocarcinoma of rectumNormal rectal tissue

0.5550.555

1.261.70

1.060 .7160 .667

0.7180.930

1.05

0.7051.16

0.8750.306

0.7160.612

1.100.983

0.7900.306

1.230 .880

1.641.74

0.8180.464

1.31

1.22

1.281.16

Pool of 10 squamous cell carcinomas of lungPool of 10 normal lung tissues

Neuroblastoma of lungNormal lung tissue

7330 1 130

7320± 190

6098 ± 445652± 1396240 ± 71

6647± 59

6670 ± 200

6340 ± 30

6380± 500

6120± 4905320± 140

6520± 2706810 ± 110

7250 ± 150

6422± 2646539 ± 66

1.38

1.060.7300.682

0.6560.930

1.12

1.591.32

0.760

1.141.16

2.002.06

1.851.70

1.831.901.84

1.811.76

1.86

1.901.80

1.891.88

L831.85

1.841.85

1.881.80

1.881.88

1.841.85

1.791.50

1.90

1.82

1.77

1.81

M23@60TaM23-61N

M23-14TM23-15N

M33-106TM33-107NM33-105N

M33-102TM33-104N

Z16-119T

M23-58TM23-59N

M23-88TM23-89N

M23-100T

M23-1O1N

M33-1 lOTM33-111N

M23-98TM23-99N

M23-118T

M23-119N

M23-2OTM23-21N

Z16-44T

Z16-62N

Z16-36T

Z16-94T

M47-24TM47-23N

Teratoma of ovaryNormal ovarian tissue

a N, normal tissue; T, neoplastic tissue.

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Cancer Research Vol. 25, September 19651246

a high of 2.06, with most values close to the mean of 1.84(the corresponding value for the calf thymus DNA standard was found to be 1.93). Considering the small degreeof protein contamination and the limited amount of mostsamples, direct determination of protein content was notfeasible. On the basis of the absorbance ratios, sampleNo. Z16—62Nappears to have the most protein contamination and also was the only sample whose ultraviolet absorbance data differed significantly from its neoplastic

tissue pair.The nature of the source of tissues used in this study

severely limited the amount of samples available. Consequently, it was not pos.sible to perform all of the tests inevery case. An attempt was made, however, to performthe same studies with normal and neoplastic tissue DNAfrom the same patient. In the tables, the letter “N―appearing after the laboratory number indicates normaltissue, and “T,―neoplastic tissue. Data on tissue pairsappear in the tables immediately below each other.

c) Thermal hyperchromw effecls.—Theincrease of ultraviolet absorbance of heated solutions of DNA was determined in a manner similar to the procedure outlined byMarmur and Doty (25, 26). The melting profiles of DNAin solutions of 0.15 M NaCl and 0.015 M Na-citrate (pH7.0) were measured with a Beckman DU spectrophotometer, which had been fitted with thermal spacers for heating arid cooling by circulating liquids. A small superstructure was constructed to fit over the cell compartmentto provide a means of positioning the cell holder withoutremoval of the thermometer. The temperature was increased at the rate of about 0.1°/mm starting at about75°C. Ground glass-stoppered cells were used for allmeasurements reported, and the results have been corrected for thermal expansion of the solutions.

d) Light scattering.—Thelight scattering measurementswere made with a Brice-Phoenix Light Scattering Photometer. A Universal Model 1000 was used in the earlyphases of this work,3 and Model 2000 was used in thesecond half of the study. The instruments were calibrated by use of an opal glass standard according to themethod of Brice et al. (5, 6). With the use of the instrument constant f3obtained by this procedure, an R90 valueof 44.4 X 10—scm' at 4358 A for benzene was obtainedwith the Model 1000, compared with the value of 48.8 X10—6cm' obtained with the Model 2000. Both valueswere measured at 25°C,with an empty cell used to correct for the background [comparable values in the literature (23) range from 44 X 10@ cm' to 50 X 10@ cm'].The difference between these values is probably due in partto the variation of the level of stray light between the 2instruments; this may not be a factor in the measurements

of DNA solution against their buffer blanks. The scattering wa.s measured in the angular range of 25°—135°withthe use of both cylindrical and hand-blown conical cells.The cells were calibrated against a square cell and checkedfor optical homogeneity with solutions of fluorescein. Thelight scattering measurements were carried out at roomtemperature, with 436 mj@ light isolated from an AH-13

mercury vapor lamp, with a blue filter combination.

3We are grateful for the loan of this instrument to Dr. R. Milton,Research Department of the Linde Co., Division of Union Carbide Corp., Buffalo, N. Y.

The solutions of DNA were clarified by centrifugationat 25,000 X g for 2 hr in a refrigerated centrifuge (3°—4°C)at a concentration of 1 mg/ml or less. The buffer solutions were first filtered with Parti-Cell membrane filtersand then centrifuged in the same manner. Only the top@volume was used for preparation of solutions for lightscattering measurements. The cells and pipets wererinsed with double distilled water that had been clarifiedin the same manner as the buffer. Weight averagemolecular weights (M) and radii of gyration (R0) werecalculated by the method of Zimm (35), by the relationship:

Kc 1( ‘6@@D222

@;-= M1@1 + 3X2 “°sin2 0/2 + . . .} + 2A2Q(O)c

in which K is an optical constant dependent on the refractive index increment of the DNA solution, c is the concentration of DNA, R8 is the Rayleigh ratio (reduced intensity) for excess scattering at the angle of 0, Xo is thewave length of the incident light in vacuum, n0 is the refractive index of solvent (buffer), 0 is the scattering angleand A2 is the second virial coefficient. This last term couldbe neglected since the Kc/Re ratio was found to be independent of the concentration in all cases tested with abuffer of 0.15 M NaCl and 0.015 M Na-citrate at pH 7.0and up to a maximum DNA concentration of about 0.2mg/ml (i.e., the “crossbars―of the Zimm-plot werefound to be parallel to the sin2 0/2 axis).

R0 was determined from the ratio of galvanometer readings4 at angle 0 to zero angle (G8'/G0') corrected for the

blank (G8/G0), by multiplication with the polarizationfactor (1/1 + cos2 0), the instrumental calibration constant fi (see above), and a correction factor (sin 0) for thescattering volume as viewed by the photometer:

$ sin 0 IG.'

R0 =@@@@

G0

A program was written for an IBM 1620 computer basedon the expression:

r 2 2 21fi i$l6irnoRa i . 2

ze = kM@ [ KM3X02 _Jsin 0/2

where Z6 represents fic/Re.The program was designed to accept the galvanometer

readings of the solution and blank and to give a “leastsquares― intercept of Ze versus 5j@20/2. The weight average molecular weight of the DNA is obtained from theintercept:

Al- KZ8...0

and the radius of gyration from:

R0 ( slope 3@2 \112intercept@ 16w@no2)

Corresponding values for the particle scattering factors[P(0)] contour length (L), rod length (ir), persistencelength (a), and “stiffness―factor (x) were calculated fromthe above parameters in the usual manner (15) (see alsofootnotes to Table 6).

4 Corrections were made for the absorption of the filters.

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SampleBufferdn/dcWorthington

calf thymus, Lot599Tris0.182Worthingtoncalf thymus, Lot602Saline-citrate0.177Worthingtoncalf thymus, Lot602Saline-citrate0.177M23-14T,@

medium differentiatedadenocarcinoma ofstomachSaline-citrate0.198M23-15N,

normal stomachtissueSaline-citrate0.205M23-20T,neuroblastoma of lungSaline-citrate0.203

DeterminationS madeNo.DiagnosiSTm (@C)Hyperchromiceffect(%)FreshM33_106T0

M33—107NM33—105NM33—104NM33—11OTM33—111NM47—24TM47—23NAdenocarcinoma

of colonNormal colon tissueNormal colon mucosaNormal cecal mucosaMedium differentiated adenocarcinoma of rectumNormal rectal tissueTeratoma of the ovaryNormal ovarian tissue85.8

86.385.38,5.386.386.385.486.049

484847494737

41After

storageM23—58TM23—59NM23—88TM23—89NM23—118TM23—119NM23—20TM23—21NAdenocarcinoma

of rectumNormal rectal tissueMedium differentiated adenocarcinoma of the rectumNormal rectal tissuePool of 10 squamous cell carcinomas of lungPool of 10 normal lung tissuesNeuroblastoma of lungNormal lung tissue—‘

83.085.284.285.085.484.483.0—“

502856444653

50FreshLot

602Worthington calf thymus86.944

FIEL et al.—Biochemical Parameters of Neoplasia 1247

e) Refractive index incrernent.—Therefractive index increment of DNA solution was measured at room temperature with a Brice-Phoenix Differential Refractometerwith 436-mj@ light isolated from an AH-13 mercury vaporlamp. The instrument was calibrated with aqueous solutions of sodium chloride. The refractive index incrementwas measured on 3 samples of calf thymus DNA and 3samples of human DNA. The results are shown in Table2. The value of 0.177 was used to calculate the molecularweights of calf thymus DNA, and the average value of0.202 was used in the calculation of molecular weights ofall human DNA samples.

f) Intrinsic viscosity.—Viscositymeasurements weremade at 37°Cwith a Ubbelohde, 4-bulb viscometer. Themean shear rates for the bulbs were calculated from the

Koepelin formula (22) and found to be approximately 100sec@, 50 sec1, 30 sec1, and 15 sec', repsectively.

RESULTS

a) Hyperchromic effect.—Table 3 lists the Tm and %hyperchromicity values of the various samples tested.

TABLE 2REFRACTIVE INDEX INCREMENT (dn/dc)

The Tm value of 86.9°Cfor calf thymus DNA correspondsto the value 87.0°C reported in the literature (26). Tmmeasurements of some samples shown in in Table 3 (asindicated) were made several months after the DNA wasisolated and otherwise evaluated. In the case of 1\123-20T and I\123-21N, the determination was done 1 yearlater. This may account for the seemingly low meltingtemperature. Although the samples had been stored in afrozen state, it is likely that some degradation had occurred. The melting profile of M23-58T indicated severedenaturation, and that of M23—88T, although exhibiting atypical transition curve, had a total hyperchromicity ofonly 28 %. The remainder of the values were all determined from fresh samples. The results are characterizedby narrow transition curves with an average Tm of 85.8°C.The increased thermal stability of the DNA from somemammalian tissues noted by Savitsky (30) was not apparent here. It appears that Tm values of DNA preparations determined from fresh samples fall into a relatively

narrow range. There is no significant difference in thisrespect between normal and neoplastic tissue. There isno indication of denaturation or loss of the double-strandedhelical structure.

b) Light scattering studies on calf thyrnus DNA .—Acommercial calf thumus DNA preparation (Worthington Biochemical Corp.) was chosen to serve as reference for themolecular weight determinations. The results of themolecular weight measurements of this material areshown in Table 4, along with those of our own calf thymusDNA preparation made by the modified (1) Marmur (24)method of isolation. Chart 1 illustrates a typical Zimmplot used in these calculations. The values of the molecular weights of the samples listed in Table 4 fall approximately in the middle of a rather wide range of values thathave been reported for calf thymus (15, 16). Chart 2 illus

TABLE 3

a T, neoplastic tissue; N, normal tissue.

MELTING PROFILE OF DNA PREPARATIONS

a T, neoplastic tissue; N, normal tissue.b Could not be determined because of severe denaturation of sample.

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No.PreparationBufferrnode1No.@edM X lO@R0(A)1

23456Worthington,

Lot 599Worthington, Lot 599Worthington, Lot 602Worthington, Lot 602Worthington, Lot 602Marmur preparationTris

Saline-citrateSaline-citrateSaline-citrateSaline-citrateSaline-citrateBP1000

BP1000BP1000BP2000BP2000BP10004.88

5.844.675.165.346.102600

31302840281030001840

20 /7/7/

40

30

P(e)_I 20

I0

0

THEORETICAL COlL#@

U@

—@@ x. 25

@@@@ THEORETICALRODSI 2@O 40 ‘ @O 80 00

h2 RG2

No.DiagnosisM X 10'R@(A)Zl6@@36TaPoorly

differentiated squamouscell carcinoma of maxillarysinus20.53300Z16—44TNeuroblastoma

of adrenal, mediumdifferentiated28.64500Z16—62NNormal

adrenaltissue>60.0—Z16-94TSpleen,Hodgkin's disease, and hy

persplenism50.74000

1248 Cancer Research

TABLE 4

Vol. 25, September 1965

MOLECULAR WEIGHTS (M) AND RADII OF GYRATION (R0) OF CALF THYMUS DNA PREPARATIONS

r.- 160

‘C

@I2

‘5

CHART 2.—Reciprocal particle scattering factors. Worthington calf thymus DNA (Lot 602, experiment 5) in 0.15 M NaCl +0.015 MNa-citrate buffer, pH 7.0.

TABLE 5MOLECULAR WEIGHT (M) AND RADIUS OF GYRATION (R0) OF Hu

MAN DNA PREPARATIONS MADE BY THE ZAMENHOF METHOD

I I I I I I I I I

0 0.2 0.4 0.6 0.8Sin2 8/2 + I x 104C

CHART 1.—Zimm plot. Worthington calf thymus DNA (Lot602, experiment 3) in 0.15 M NaCl + 0.015 M Na-citrate buffer,pH 7.0. DNA concentrations of 15.6 X lOs, 30.7 X 106, 45.4 X106, 59.6 X 10—sgm/ml.

trates the experimental scattering envelope of the Worthington calf thymus sample (Lot 602) compared with thetheoretical curves of a coil with a varied degree of stiffness.The stiffness factor (x) for this calf thymus sample has beencalculated from an approximate persistence length [i.e.,with only the first term of the expression (15)] and foundto be 29.5. The scattering envelope of the DNA lies betweeti the extremes of a theoretical coil and rod, as described previously by other investigators (11, 17). In thisinstallce the experimental points are closer to the rod formthan to the coil. This rigidity is also apparent from theviscosity studies (see Table 9) on another preparation fromthe Worthington calf thymus sample (Lot 602), which hada similar stiffness factor (x = 24) . In a single experimentDNA was extracted from fresh calf thymus by a modifiedZamenhof procedure, and the molecular weight was foundto be too high for accurate determination. This was probably the result of a gel structure or aggregation (8, 9).

c) Light scattering studies on human DNA prepared bythe Zainenhof method.—Table 5 lists the molecular weightand radius of gyration for 4 samples of DNA prepared fromhuman tissue with the Zamenhof procedure (34). The 2ndand 3rd samples represent a matched pair; that is, thesample of normal tissue and the sample of neoplastic tissuewere taken from the same patient.

a T, neoplastic tissue; N, normal tissue.

Although the molecular weights obtained for thesepreparations seem unusually high, a consideration of thetheoretical restrictions implicit in an extrapolation for thecase in which R0 > 0.5X (29) places these values in thecategory of minimum molecular weights, particularly inview of the recent results of low-angle measurements reported by Froelich et al. (14). A typical Zimm plot forthis series is presented in Chart 3. In addition to the considerations mentioned above, it is also apparent thatthere is a high degree of inherent uncertainty in extrapolating such a large slope to a zero abscissa. Chart 4represents the scattering envelope of sample Z16-91T.The molecular weight of 50.7 X 106 as stated in Table 5was obtained from a linear extrapolation from 90°to 25°.It is apparent from Chart 4 that a large error is involved,since this would require an upward curvature below 30°(as indicated). Since 25°is our experimental limit it isnot possible to determine whether an upward curvature

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FIEL et al.—Biochemical Parameters of Neoplasia 1249

neoplastic rectal samples are considered, a mean molecularweight of 1.50 X 106 is obtained with a standard deviationof 0.087 X 106. When all 7 adenocarcinomas are considered, a mean value of 1.52 X 10@is obtained with astandard deviation of 0.278 X 10g.

Table 7 summarizes mean values and standard deviations for the physical characteristics of DNA moleculesfrom neoplasias compared with those from correspondingnormal tissue. Significant differences (P < 0.005) are seenbetween the tumor and normal samples in molecularweight, contour length (L) and the stiffness factor (x).It appears that DNA from neoplastic tissue is a somewhatsmaller and more rigid molecule than that from corresponding normal tissue.

It can be seen from Table 6 that in the case of I\I33-106,-107, -105 actually 3 samples were available : aderiocarcinoma of the colon, normal colon, and normal colonmucosa. It was throught that normal colon might notnecessarily be an adequate control for the tumor, since thelatter is of epithehoid origin, where the majority of the elements in the former are not. For this reason normal colonmucosa was added as an additional control. For the purpose of this evaluation this tumor sample was comparedseparately with each of the control tissues. Thus calculations were based on 12 pairs of tissue, although only 23tissue samples were analyzed.

The justification of grouping different types of tumorstogether for the purpose of evaluation may be open toquestion. Adenocarcinoma is the only histologic typefrom which reasonable numbers are available. Table 8evaluates differences between the aderiocarcinomas and thecorresponding normal tissues. The conclusions are thesame as for all tissue pairs pooled.

Only 1 exception is noted from the smaller molecularweight of neoplastic DNA : in a pair of samples from anovarian teratoma and the corresponding normal ovariantissue. Two exceptions appear from the increased stiffness of tumor DNA: in the case of the same pair of samplesfrom the ovarian teratoma and normal tissue, and in the

case of pooled samples of squamous cell carcinoma of theJung compared with normal lung tissue. These findingsmay be due to the polydispersity of the DNA in the pooledsamples or in the multiple tissue types of the teratoma.

40

30

@cor' 20

CHART 4.—Reciprocal particle scattering factors. SampleZ16-94T, spleen, Hodgkin's disease, and hypersplenism, in 0.15M NaC1 + 0.015 M Na-citrate buffer, pH 7.0.

.

0

0.2 0.4 0.6

Sin2 8/2 + 5xIO3C

0.8

CHART 3.—Zimm plot. Sample Z16-44T, medium differentiated neuroblastoma of adrenal, in 0.15 M NaCl + 0.015 M Nacitrate, pH 7.0. DNA concentrations of 36.2 X 10@,71.8 X 106,87.8 X 10—s,103 X 106 gm/ml.

is present. In view of an observed downward curvaturebelow 60°,it seems likely that the intercept has been overestimated.

d) Light scattering studies on human DNA isolated by themodified Marmur method.—The modified (1) Marmurmethod of isolation was chosen as the general procedurefor this work on the basis of reproducibility and yieldThe results of light scattering studies on the DNA preparations made by this procedure are shown in Table 6. Thetable headings correspond to weight average molecularweight (M), the radius of gyration (R0), the contourlength (L), the rod length (ir), the persistence length (a),and the stiffness factor (x) (15). Definitions and principles of calculation are given in the explanation to theheadings of Table 6. Chart 5 represents examples of theexperimeiital values of the reciprocal particle scatteringfactor [P(0)'J for some of the samples, accompanied bythe corresponding theoretical curves for rod- arid coilshaped molecules.

In examining the results listed in Table 6, it is apparentthat the molecular weights arid radii of gyration are withinthe rather broad range of those values reported in theliterature for chicken erythrocytes, rat liver, salmon sperm,and the many other sources of DNA (15) . Twelve of the23 samples have molecular weights within the limit of1—2.4X 106 given by Cavalieri et al. (9) for DNA fromdiverse sources, using deproteinization with chloroform andoctanol. Four other samples had molecular weights between 2.4 and 3.4 X 10@and the remaining 6, between 3.4and 10 X 10―. It is interesting to note that many of thelow molecular weight samples (M < 3.4 X 10@)appear tobe very highly extended, as indicated by a large length-tomass ratio. This is apparent from the similarlty of therod length (lr) to the corresponding contour length (L) andfrom the relatively small x values.

In view of the past inconsistencies found in the molecular weights reported from a single tissue, notably calfthymus (probably due in part to variations in the isolationprocedure by various investigators), it is not surprisingthat we have obtained a fairly wide range when using avariety of tissues. In some instances, however, the rangeis found to be quite narrow. For example, if only the 4

I0

h2 RG2

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No.DiagnosisM° X lO'R0 (A)bL (A)c1,.(A)'@a6x'M23-60T@

M23-61NAnaplasticcarcinoma of stomach

Normal stomach tissue2.79 8.352,470 1,69014,40043,0008,560 5,8601,270 19911.3216M33-106T

M33-107NM33-105NAdenocarcinoma

of colon

Normal colon tissueNormal colon mucosa1.81

6.597.191,470

2,4001,8009,340

34,00037,0005,090

8,3206,230694

50826313.5

67.0141M33-102T

M33-104NUndifferentiated

carcinoma of cecum

Normal cecalmucosa2.19 3.172,2901,97011,30016,3007,9306,8201,3907148.1322.8M23-58T

M23-59NAdenocarcinoma

of rectumNormal rectaltissue1.62 5.921,5304,0308,33030,5005,30013,900835 1,6009.9819.1M23-88T

M23-89NMediumdifferentiated adenocarcinoma of rectum

Normal rectal tissue1.41 2.302,300 1,6207,250 11,9007,970 5,6202,200 6613.3018.0M23-100T

M23-1O1NMedium

differentiated adenocarcinoma of rectumNormal rectaltissue1.49 3.331,8902,5807,67017,2006,5508,9401,4001,1665.4814.8M33-11OT

M33-111NMedium

differentiated adenocarcinoma of rectumNormal rectaltissue1.49 1.b41,6601,2907,6707,9405,5504,4701,078 6297.1212.6M23-98T

M23-99NMedium

differentiated adenocarcinoma of lungNormal lung tissue1.00 1.801,530 2,2105,160 9,2905,300 7,6601,360 1,5803.795.88M23-118T

M23-119NPoolof 10 squamous cell carcinomas of lung

Pool of 10 normal lung tissues4.69 5.221,800 2,70024,20026,9006,240 9,350402 81360.233.2M23-20T

M23-21NNeuroblastomaof lung

Normal lung tissue3.27 9.722,270 3,32016,80050,1007,860 11,500918 66018.375.9M47-24T

M47-23NTeratomaof ovary

Normal ovarian tissue1.60 1.171,920 1,6108,260 6,0506,650 5,5501,340 1,2966.16 4.67

1250 Cancer Research Vol. 25, September 1965

e) Viscosity.—The Worthington preparation of calf (12), this gives a molecular weight of 4.4 X 10@,which is inthymus DNA (Lot 602) was found to have an intrinsic vis- fair agreement with the light scattering data (Table 4).cosity of 39.6 dl/gm. With the use of the relationship However, there is no generally valid relationship estab[‘1]= KM''2 experimentally derived for calf thymus DNA lished between the viscosity and molecular weight of DNA

TABLE 6MOLECULAR PARAMETERS OF DNA ISOLATED BY THE MODIFIED MARMUR METHOD FROM NORMAL AND

NEOPLASTIC HUMAN TISSUE

a Weight average molecular weight (M) is given by

Egi M@M=t

@:gi

in which gj is the amount in grams of the components with molecular weights of M@.b The radius of gyration (H0) is given by

R0 (>Jm@rj2/@m@)U2

where rn is the mass of the ith element located at a distance r@from the center of mass. This parameter, in conjunction with Al, refleets the average shape of the molecule.

C The contour length (L) is the end-to-end distance of a fully extended molecule having a molecular weight of M and the linear

molecular weight density of 194 A' calculated for the Watson-Crick model.d The rod length (ir) @8the end-to-end distance of a rodlike molecule having a radius of gyration equal to RG . It is related to the

radius of gyration by the expression B02 = l@2/l2. For a perfect rod, Z@= L.e Persistence length (a) is the sum of the projections of the average orientations of all segments of a chain on the direction of the

1st chain element. For a rod, a = ir ; for a coil, a is equal to the length of an average segment.I The stiffness factor (x) is equal to the number of persistence units in a chain. x = L,fa. For a perfect rod, x = 1; the value

increases with increasing compactness (decreasing “stiffness―).9 T, neoplastic tissue; N, normal tissue.

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MEASUREMENT―@a

:@ @MEAN

VALUE ± STANDARDDEVIATIONPTumorNormalM

x 10@

B0 (A)L (A)11. (A)a

x12

1212

1212

122.10

± 1.03

1,883 ± 36710,810 ± 5,305

6,507 ± 1,2841,131.7 ± 471.6

13.4 ± 15.44.69

± 2.87

2,268 ± 79524,181 ± 14,801

7,854 ± 2,743836.2± 471.152.6 ± 64.9<0.01

<0.01

<0.05

MEASURE‘CENT―No.

orPAIRSMEAN

VALUE ± STANDARD DEVIATION

@_______________TumorNormalM

x 10-6R0 (A)L (A)ir(A)ax7

7

77

7

71.52±0.278

1,693±305

7,822±1,433

5,835±1,067

1,180.1±535.9

8.1±4.34.09±2.406

2,276±896

21,118±12,383

7,877±3,083

915.3±534.1

39.8±49.0<0.05

<0.05

<0.05

FIEL et al.—Biochemical Parameters of Neoplasia

CHART 5.—Reciprocal particle scattering factors of human DNA prepared by the modified Marmurmethod.

1251

P(s)-I

•o 0 8

htR8t—

TABLE 7COMPARISON OF PHYSICAL CONSTANTS OF DNA FROM

NORMAL AND NEOPLASTIC TISSUE PAIRS

TABLE 8COMPARISON OF PHYSICAL CONSTANTS OF DNA FROM

ADENOCARDINOMAS AND CORRESPONDING NORMAL TISSUE

a See Table 6 for definitions of abbreviations. a See Table 6 for definitions of abbreviations.

from different sources. Even in the case of various calfthymus DNA preparations, the above relationship is notvery well satisfied by a substantial portion of the 34 different samples listed by Geiduschek and Holtzer (15). Itwas suggested by Spitkovskii ef al. (31) that the proportionality factor (K) varies with the configuration of themolecule. These authors used the expression [@J= KM.

The results of our viscosity measurements on 4 pairs ofDNA preparations from normal and malignant tissues ofhuman patients are shown in Table 9.

The 4 tumor DNA samples seem to satisfy the 2nd ofthe above expressions remarkably well; within this admittedly small series, the intrinsic viscosity appears to beproportional with the 1st power of the molecular weightas determined by the light scattering method (Table 6).The corresponding 4 normal DNA samples, on the other

hand, do not show the same linear relationship betweentheir intrinsic viscosities and molecular weights; theirrelatively low viscosities may be related to their significantly higher x values (more compactness), apparent fromthe light scattering measurements (Table 6), and may beindicative of aggregation.

For further corroboration of the values obtained by thelight scattering method, the radii of gyration were calculated from the intrinsic viscosity measurements : (a) for arandom coil, with the Flory-Fox relationship (13) according to Tanford (33); and (b) for a rodlike model, on thebasis of the equation of Sadron (29). The values so obtainedfor the 8 DNA samples for which viscosity data are available are given in Table 9 as R0 (vis-coil) and R@ (vis-rod),respectively. Comparison of these values with the radiiof gyration (R0) obtained by the light scattering method

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SampleViscosity [,;](dl/gm)R0

(viscoil/iR0

(visrod)Calf

thymus DNA, Worthington, Lot60239.618513088M23@60T,b

anaplastic carcinoma of stomachM23-61N, normal stomach tissue47.0 18.01649172728872800M23-58T,

adenocarcinoma of rectum

M23-59N, normal rectal tissue40.0 60.01300212421723816M23-88T,

medium differentiated adeitocarcinoma of rectum

M23-89N, normal rectal tissue24.5 16.01053107817601792M23-20T,

neuroblastoma of lungM23-21N, normal lung tissue60.0 40.01890236432203920

1252 Cancer Research Vol. 25, September 1965

TABLE 9

INTRINSIC VISCOSITYOF DNA SAMPLEStoma of ovary) and M47-23N (normal ovarian tissue),the only DNA pair that originally showed a reverserelationship (see Table 6). After standing for 2 weeks at4°Cin a stock solution at an approximate concentrationof 1 mg/mi, sample M47-24T was found to have a molecular weight of 1.67 X 10@ (compared with a previousvalue of 1.60 X 10@), whereas the corresponding normalsample M47-23N had increased from 1.17 X 10@to aminimum value of 1 .7 X 10@ (a fairly large dispersity ofpoints did not permit a precise evaluation).

There are some indications [Srinivasan et al. (32)] thatDNA from neoplastic tissues contains a higher proportionof “abnormally―methylated purine and pyrimidine basesthan normal DNA. Steric hindrance as well as hydrophobic forces due to the methyl group would be expectedto interfere with coiling and would tend to increase therigidity of the molecule.

There is, of course, no evidence to indicate that thedifferences noted reflect an in vivo difference between

normal and neoplastic tissue DNA. Although circumstantial evidence, such as abnormalities of some neoplastic chromosomes and possible differences betweenmitotic activities of normal and neoplastic cells, may leadus to suspect that physical differences may exist, we haveno indication as to what extent they may be manifested atthe molecular level.

Our data indicate that the Zamenhof procedure of isolation may lead to DNA preparations of relatively highmolecular weight, perhaps as the result of incomplete

deproteinization. In contrast to this, the ?tlarmurprocedure, which makes extensive use of chloroformisoamyl alcohol as a deproteinizing agent, generally leadsto lower molecular weights. These lower molecularweights are in accord with the values obtained by Cavalieri(9) with chloroform-octanol used as the deproteinizingagent. In both procedures, erizymic degradation of DNAwas prevented by the use of EDTA and citrate, and carewas taken to keep the extent of shear during homogenization at a constant level to eliminate the possible effect ofmechanical degradation. The wide divergence of valuesobtained by the 2 methods serves as dramatic evidence ofthe dependence of molecular weight on the isolationprocedure used. However, the fact that a narrower rangeof values can be obtained with a particular procedure doesnot imply that these are necessarily more representative ofthe in vivo functional state of these molecules.

Since DNA preparations obtained from the same tissueby either of the 2 isolation procedures showed no difference in their priming activities (1), we may conclude

that there is no simple correlation between the primingactivities and molecular weights of the DNA preparationsstudied.

ACKNOWLEDGMENTS

We wish to acknowledge the devoted technical assistance of I.Csaba, J. Hahn, I. Mink, and A. Penny and of our research nurses,N. Edwards, R. Markus, V. Pawlak, and B. Striegel. We aregrateful to Dr. J. W. Pickren for his help in the examination ofhistopathologic preparations and to Dr. R. K. Ausman and R.Edie for their assistance in writing and executing the computerprogram. We are indebted to Dr. K. Paigen, Dr. H. Weinfeld,and Dr. G. Markus for critically reading the early drafts of thismanuscript.

a@ radius of gyration.

b T, neoplastic tissue, N, normal tissue.

(Table 6) shows that the latter fall generally between thecorresponding values of coil and rod calculated from theviscosity measurements. This is in agreement with the“scattering envelopes― of these samples (Chart 5) inwhich the experimentally determined particle scatteringfactors are shown to fall between those of a theoreticalcoil and rod, respectively. Thus, the description of theDNA molecule as a “stiff―coil (11) seems to be validfor these DNA preparations from human tissues.

DISCUSSION

Radii of gyration calculated from light scattering andviscometric studies seem to indicate that DNA from

normal as well as from neoplastic human tissue fallsbetween the values established for a model random coiland a model rod-shaped molecule. This result is generallysupported by the “scattering envelopes― [P (0)' curves]obtained for these samples. The DNA molecules maythus be described as “stiffcoils.― It appears that themolecular weight, size, and shape of the DNA moleculeextracted from human tissue is similar to the DNA isolatedfrom animal tissues, as well as from a variety of other

sources, such as Escherichia coli, pneumococcus, and

salmon sperm (15).DNA isolated from neoplastic tissues seems to be of

smaller molecular size and more rigid (less compact) thanDNA from the corresponding normal tissues. Althoughit is tempting to speculate on the possible biologic significance of this difference, if any, this must be done with

considerable caution. One explanation may be that thedifference in molecular weights between the normal andtumor DNA is a result of a difference in the response of aspecific tissue to the isolation procedure (18). It ispossible that DNA in normal tissue is somehow protectedand less prone to break down during extraction. It isalso possible that the normal tissue DNA has a greatertendency to aggregate than its neoplastic counterpart.Some indication of the latter possibility has been seen onrepeating the measurements on samples M47-24T (tera

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FIEL et al.—Biochemical Parameters of Neoplasia 1253

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1965;25:1244-1253. Cancer Res   R. J. Fiel, T. J. Bardos, Z. F. Chmielewicz, et al.   Neoplastic Human TissueProperties of Deoxyribonucleic Acid from Normal and Biochemical Parameters of Neoplasia: II. Some Macromolecular

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