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Polycyclic hydrocarbon-induced rat sarcomas correlated to disturbances in the deoxyadenylate regions of the tumor DNAs
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Polycyclic Hydrocarbon-Induced Rat Sarcomas Correlated to Disturbances in the Deoxyadenylate Regions of the Tumor DNAs* RONALD W. PERO,~ TOMAS BRYNGELSSON,+ CHRISTINA RUDDUCK+ and GORAN LEVAN++ t Nucleic Acid Biochemistry and +Institute of Genetics, Lund University, Wallenberg Laboratory, Fack 7031,220 07 Lund, Sweden A b s t r a c t - - T e n separate groups containing a total of 51 rat sarcomas induced by the polycyclic hydrocarbons were examined for inducing agent-related abnormalities in their DNAs. The levels of deoxyadenylate (dA) regions in normal and tumor rat DNAs were estimated from annealing [3H]-polyuridylic acid to D N A and treatment of the resultant hybrids with ribonuclease A. All the tumor material had significant reductions in the (dA ) regions of their DNAs when the levels were compared to normal rat DNA. Although the (dA) reductions were rather heterogeneous from one tumor group to the other no matter the inducing agent, the greatest decreases tended to be found with tumors induced by 20-methylcholanthr ene (M C ) and 7,12-dimethylbenz(a)anthracene (DMBA). No relationship was found between (dA) alterations and the types of chromosomal aberrations in the tumor cell populations. However, normal and tumor DNAs, fractionated into A + T rich and G + C rich D N A by thermal elution from hydroxylapatite columns, showed specific neoplastic-associated (dA ) disturbances in the A + T rich D N A of tumors induced by 3,4-benzopyrene (BP), M C and D M B A . The base compositions of the tumor material were also analyzed and compared to normal rat D.\:t. Tumors induced by D M B A showed highly significant decreases in ~o A + T ~'hereas B P and M C induced tumors tended to show only small increases in o i) A + T. Some tumors induced by BP, M C and D M B A were unaltered in base composition. Together our results are consistent with the hypothesis that D M B A is the strongest carcinogen in the series because it interacts preferentially with A + T rich DNA, which is the type of D N A most important for polycyclic hydrocarbon-induced neoplasm in the rat.
INTRODUCTION
other polypyrimidine stretches were approximately 25-200 nucleotides in length [2-4, 6, 10] and were concentrated into repeated sequences in the D N A [4, 7, 11]. The fact that these oligohomopolymeric rich regions could not have arisen from a random selection model [1], together with their specificity for location into repeated DNA, have strongly suggested a biological function for these regions. These observations are further supported from similar work on nRNA. It has been shown that oligo(U) and oligo(A) regions of approximately 25-35 nucleotides are a normal component of n R N A [12-14] indicating that their template for transcription must have been the (dA) or (dT) i'egions in the DNA [13]. The oligo (U) regions in n R N A have also been shown to be located primarily in the
THE PRESENCE of (dA) rich stretches and polypyrimidine runs in eukaryotic D N A has now been validated by several laboratories [1-11]. The (dA) or (dT) rich sequences and
Accepted 12 January 1978. *This work was supported by the Swedish Cancer Society (RMC 76:101) and by a special grant to the Dalby Community Care Sciences program from the National Board of Health and Social Welfare in Sweden. The abbreviations used are: (dA), deoxyadenylate; (dT), deoxythymidylate; (U), ribouridylate; (A), riboadenylate; BP, 3,4-benzopyrene; MC, 20-methyleholanthrene, DMBA, 7,12-dimethylbenz(a)anthracene; SSC, 0.15M NaCI+0.015M sodium citrate, pH=7.2; Txl, temperature at which 50% native DNA becomes single stranded nRNA, nuclear RNA. 961
9~
Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and G6ran Levan and not karyotyped. The groups of tumors designated DMBA-17 and DMBA-19 were transplanted for a single passage in newborn Wistar-Furth rats as described elsewhere [23]. All tumors were frozen and stored at - 7 0 ° C until used for DNA extraction. The livers, spleens, inner skin covering the abdominal cavity and full term embryos were dissected from adult Wistar-Furth rats and either processed directly or stored at - 7 0 ° C . These tissues were taken from apparently healthy animals and served as the control sources of normal rat DNA.
5'-end of n R N A which is mainly transcribed from repeated DNA template [14, 15] and confirms the location of (dA) clusters there. Since the 3'-end of n R N A contains unique DNA transcript or the mRNA, then the implications are that (dA) transcripts or the oligo(U) regions might play ~ role in the proposed processing of n R N A into m R N A [16]. At any rate, the exact function of (dA) transcripts is still unknown although some biological function most certainly can be implied. Our laboratory has assumed an important function for the (dA) regions in either the transcription or processing of genetic information, and has attempted to assign a role to them in neoplasm. Our results have shown that (dA) alterations could be demonstrated in the DNA from human breast tumors [17], human CLL lymphocytes [18] and rat tumors induced by Rous sarcoma virus or DMBA [19]. Generally, the more malignant cell types were associated with reduced levels of (dA) regions in their DNAs. Although (dA) disturbances were found in both A + T rich and G + C rich DNA [17], some specificity of change was observed for both main band and low repeated DNAs (18). However, our ability to demonstrate specific (dA) alterations common to neoplasm has been complicated by looking at neoplasms where the oncogenic agent is not known. Presumably, the (dA) alterations caused from viral carcinogenesis where the viral genome is involved could be quite different from those arising from chemical carcinogens. Therefore, in this study we have examined rat tumors induced by a class of closely related carcinogens, the polycyclic hydrocarbons, and where the carcinogenic potency varies in a predicted way [20, 21]. With this well defined tumor induction model, we have tried to show that tumors induced by structurally-related chemicals have (dA) alterations in specific areas of their DNA.
Extraction of DNA The rat tissues were minced in ice cold 0.1M E D T A - 0 . 1 M NaCI, pH 7.5 and homogenized at 4°C by 2 x 15-sec pulses at 1 n~lin intervals with an Ultra-Turrax. The DNA was extracted from the homogenates by the M a r m u r method [24] except for the tinal alcohol precipitation. Instead of the precipitation step, 100 #g/ml ribonuclease A (Sigma) and 100#g/ml alpha amylase (Sigma) were added to the DNA solutions and the mixture dialyzed overnight against 51 0.1 x SSC at room temperature. Final!y, the enzyme-treated DNA solutions were made 5% sodium dodecyl sulfate and precipitated with alcoholic sodium perchlorate solution as described by Wilcockson [25]. In our hands, DNA prepared in this way was essentially free from glycogen (anthrone method), RNA (orcinol method) and protein of which contamination was always less than 5% (#g protein/pg DNA, Lowry method). We have noticed in our liver preparations from unstarved animals that high concentrations of glycogen (100 #g/ml DNA solution) can occur in our final DNA preparations, if alpha amylase treatment was omitted. We have also observed that such high levels of glycogen contamination of DNA interferes with the poly(U) hybridization on filters (data not shown).
MATERIALS AND M E T H O D S Tumor induction and normal tissue Inbred Wistar-Furth rats 8-35 days of age were inoculated subcutaneously in the right thigh with 1 - 4 m g doses of BP, MC or D~IBA dissolved in 0.1 ml tricaprylin. T u m o r s developed to 100% and were harvested 6-8 weeks after they were palpable ( < 7 months). Some individual tumors were analyzed for karyotypic abnormalities by direct fixation according to Levan et al. [22]. Other tumors induced by the same carcinogen were pooled
Poly( U) annealing method We have described elsewhere [7] the essential details of our poly(U) hybridization method and the factors which limit its reproducibility. However, we have now introduced modifications which have reduced much of the variability encountered in our earlier experiments. Approximately 15#g samples of DNA dissolved in 2 x SSC were denatured by heating at 100°C for 10 rain followed by another 10 min with an equal volume of 4 M NaCI. Treating denatured DNA with hot 2 M NaCI insured retention of (dA) rich sequences
Polycyclic Hydrocarbon-Induced Rat Sarcomas and improved on selective removal of any residual contaminating protein [7]. The solutions were cooled immediately in an ice bath, loaded onto nitrocellulose filters (Millipore ®) with suction and baked in an oven for 2--4hr at 80°C. In order to improve on our yield of DNA from smaller tumors, the alcohol precipitated DNA was often collected by centrifugation. These DNA preparations tended to contain lower molecular weight DNA fragments that are normally left behind when the DNA is spooled and thus these DNA solutions retained less on the filters. Previously severely sheared DNA affected the annealing of poly(U) [7]. However, we have compared only DNAs which retained between 75-100% on the filters, and with this level of retention the estimation of (dA) regions was unaffected by the amount of DNA immobilized (data not shown). Commercial [3H]poly(U) (Miles Laboratory, 75pCi//~M monophosphate) was dissolved in 2 x SSC and adjusted to a final concentration of 6.0#g/ml with unlabelled poly(U) (Sigma). The DNA containing filters were placed in the [3H]poly(U) solution and annealed for 17hr at 25°C with continuous mixing from the aid of a magnetic stirrer. Unannealed poly(U) was removed from the filters by ribonuclease A treatment in 2 x SSC at 5 #g/ml for 6 hr at 25°C. It was important, especially in cases where many filters were treated batchwise, to have efficient stirring for both the annealing and RNAase steps if unnecessary variability was to be avoided. The details involving radioactive counting on filters, quantitative determination of DNA and estimation of (dA) "pure" sequences from the °/o poly(U) annealing to DNA after 6 hr RNAase treatment were unchanged from our earlier studies [7]. Routine poly(U) analyses were replicated 1030 times. The amount of DNA hybridizing to poly(U) was always determined on the nitrocellulose filters after RNAase treatment and scintillation counting.
TMfractionation of DNA on hydroxylapatite The TM fractionation was carried out as we have described earlier for human DNA [17] except for a few minor details. Native DNA was loaded onto hydroxylapatite in 0.01 M phosphate buffer at 60°C and the A + T rich DNA fraction eluted between 60 and 91°C in 0.12 M phosphate buffer.
Base composition analysis The method used for base analysis was the same as reported by Wang [26]. Briefly, it is
based on the fact that all the bases except adenine react with N-bromoacetamide to cause reduction in U V absorption. Standard solutions of dAMP, dCMP, d G M P and d T M P were prepared in 0.5 M H2SO4. Since at this acid concentration d A M P and d G M P were depurinated, then the molar absorbancy coefficients used to adjust the final standard concentrations to 0.05raM were as given: adenine ( E 2 7 0 = l l . 5 6 x 1 0 - 3 ) , d C M P (E270 = 1 1 . 4 4 x 1 0 - 3 ) , guanine ( E 2 v 0 = 7 . 4 x 1 0 .3 ) and d T M P (E27o=9.58 x 10-3). Mixtures of the 4 standard solutions were used to prepare a standard curve ranging from 100% A + T to 100% G + C . It was necessary in our experiments to heat the DNA solutions in 0.5M H2SO 4 for 3hr at 50°C to insure complete depurination and allow accurate estimation of base composition. Other important details such as the preparation and addition of the brominating agent and the calculation of % A + T from the u.v. absorption changes were carried out as described by Wang [26]. RESULTS
A total of 51 rat sarcomas induced by the polycyclic hydrocarbons were pooled into 10 different groups and compared to normal tissues for alterations in the (dA) regions of their DNAs. A quantitative estimation of the (dA) regions in the DNA from normal rat tissue was always the same whether the sources used were embryo, skin or liver + spleen (Table 1). A constant level of (dA) regions in normal somatic cell DNA of the rat has thus provided the analytical basis for its comparison to the (dA) levels in neoplastic DNA. All groups of tumors reported in Table 1 regardless of inducing agent had statistically significant reductions in the (dA) regions of their DNAs. The level of (dA) reductions in the tumor DNAS varied considerably from one tumor group (MC-69) to another (DMBA17), but there was a tendency for MC- and DMBA-induced tumors to have the greatest (dA) reductions (Table 1). Such heterogeneity in the (dA) alterations of DNA from tumors induced with so structurally similar carcinogens, or even when induced by the same carcinogen (i.e., MC-12 and MC-69), suggests that additional DNA template changes involving in part the (dA) regions, have accompanied those DNA changes responsible for the neoplastic event itself. One possible way to judge the degree of additional DNA changes which might occur along with those DNA changes necessary for
964
Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and Go'ran Levan Table 1. Comparison of normal tissues to polycyclic hydrocarbon-induced tumors in the rat by quantitative analysis of the (dA ) regions in their DNAs. Tumor inducing agent and identity number* Normal rat tissues embryo skin liver and spleen BP-10 BP-I 1 BP-15 MC-12 MC- 13 MC-69 DMBA- 17 DMBA-14 DMBA- 18 DMBA-19
Number of pooled tumors
% poly(U) annealing to D N A J "
Average %/tumor/agent++
---4 5 5 6 7 4 5. 6 3 6
0.147 4- 0.017 0.145 -t-0.015 0.142 ___0.023 0.12t _ 0.005§ 0,107 + 0.005§ 0.121 _ 0.019§ 0.117 4-0.008§ 0.104 4- 0.008§ 0.059 4- 0.009§ 0.134 4- 0.008§ 0.101 4-0.006§ 0.112 4-0.011§ 0.098 4-0.010§
--0.145 --0.I 17 --0.098 ---0.110
The DNAs from groups of pooled tumors and from normal tissues of the rat were extracted, immobilized onto nitrocellulose filters and annealed to [aH]poly(U) in 2 x SSC at 25°C for 18hr. Non-specific binding of [3H]-poly(U) was removed by treatment of the filters with 5/~g/ml ribonuclease A dissolved in 2 x SSC for 6 hr (for further details see Ref. 7 and the text). *BP = 3,4-benzopyrene; MC = 20-methylcholanthrene; DMBA = 7,12-dimethylbenz (a)anthracene. J'For estimation of (dA) regions divide % poly(U) value by 2. ~.%/tumor/agent represents the average % poly(U) annealing to the DNA from all the tumors induced by the same polycyclic hydrocarbon. Hence, the average (dA) alterations expected for a tumor induced by particular polycyclic hydrocarbon in question. §P<0.0005 (Student's t-test) when compared to the values ~br normal rat tissues; DMBA-14 (p<0.025); Means +S.D. are shown. polycyclic hydrocarbon-induced neoplastic t r a n s f o r m a t i o n , m i g h t be to c o m p a r e those t u m o r s w h i c h contain high frequencies of either similar or different c h r o m o s o m a l a b e r rations. I t has b e e n s h o w n [27, 28] t h a t specific c h r o m o s o m a l a b n o r m a l i t i e s are associated with t u m o r s i n d u c e d b y the polycyclic h y d r o c a r b o n s , a n d some o f these a b n o r m a lities such as the trisomy A2 can be found in t u m o r s i n d u c e d b y all 3 o f the carcinogens BP, M C a n d D M B A [22, 27]. I n T a b l e 2 we h a v e selected BP- a n d M C - i n d u c e d t u m o r s which h a d high incidences of a p a r t i c u l a r type o f c h r o m o s o m a l a b e r r a t i o n in the t u m o r cell populations. T h i s protocol has allowed us to try a n d s t a n d a r d i z e the initial a m o u n t of D N A d a m a g e inflected b y the c a r c i n o g e n at the t i m e o f t r a n s f o r m a t i o n , as evidenced by the similar c h r o m o s o m a l a b e r r a t i o n s in the s u b s e q u e n t t u m o r cell p o p u l a t i o n , a n d to test w h e t h e r these c h r o m o s o m a l similarities w e r e specific e n o u g h to reflect similar (dA) alterations. T a b l e 2 clearly d e m o n s t r a t e s t h a t a high incidence of a distinct c h r o m o s o m a l deviation such as trisomy or the presence of a m a r k e r c h r o m o s o m e in the t u m o r cell p o p u -
lation, or even the absence of a b n o r m a l i t i e s altogether, does not predict the neoplasticassociated (dA) alterations in the D N A . A p p a r e n t l y the presence of other r a n d o m c h r o m o s o m a l a b e r r a t i o n s in the t u m o r cells m u s t m a s k a n y specificity shown b y the n o n r a n d o m , distinct c h r o m o s o m a l deviations, at least, as far as a n y correlation to (dA) alterations are concerned. I n fact, s u p p o r t for this i n t e r p r e t a t i o n c a n be d r a w n f r o m the fact, as we a l r e a d y observed in T a b l e 1, t h a t again there was a r a t h e r good correlation to inducing agent; n a m e l y , M C - i n d u c e d tumors all h a d g r e a t e r reductions in the (dA) regions of their D N A s t h a n did B P - i n d u c e d t u m o r s ( T a b l e 2). Such a correlation to i n d u c i n g a g e n t would take into a c c o u n t the total altered g e n o m e a n d not just the c h r o m o s o m a l a b e r r a t i o n s w h i c h were either specific, nonspecific or absent. W e h a v e further p u r s u e d o u r efforts to distinguish the m o r e specific neoplasticassociated (dA) changes f r o m those nonessential D N A changes which m i g h t a c c o m p a n y polycyclic h y d r o c a r b o n induction of rat sarcomas, b y m e a s u r i n g the (dA) levels in A
,%5
Polycyclic Hydrocarbon-Induced Rat Sarcomas Table 2. Lack of correlation between the chromosomal aberrations in polycyclic hydrocarbon-induced rat tumors and the (dA) alterations of their DNAs Tumor inducing agent and identity number*
Chromosomal aberration specificity
% poly(U) annealing to DNA]'
Normal rat tissue BP- 1 BP-2 MC-6 MC-7 BP-3 MC-8 BP-4 MC-9 BP-5
normal normal trisomy A2 trisomy A2 trisomy A2 marker i(2) marker i(2) marker t(3;?) marker t(3;?) marker t(11;?)
0.145 _+0.019 0.101 _+0.017 + 0.134_+ 0.017++ 0.055 _+0.002+ 0.056 _+0.003++ 0.134_+0.011++ 0.070 -t-0.002++ 0.118 +0.017++ 0.054-t-0.017++ 0.111 -+0.018++
The DNAs from individual rat tumors which had been karyotyped for any specific chromosomal abnormalities and from normal rat skin and liver were extracted, immobilized onto nitrocellulose filters and annealed to [3H]-poly(U) for estimation of (dA) clusters (for further details see Ref. 7 or text). Additional details of the chromosomal aberration designations are found in Ref. 27. *BP=3,4-benzopyrene; MC=20methylcholanthrene; The identity numbers in this table correspond to the following identification numbers in Ref. 27 presented in order from top of table to bottom: BP10, BP12, MC8, MC1, BP9, MC2, BP2, MC2+5, BP4. ]'For estimation of (dA) regions divide % poly(U) value by 2. ,*P<0.05 (Student's t-test) when corripared to value for normal rat tissue; Means _+S.D. are shown.
+ T rich and G + C rich subfractions of the native tumor DNAs. Table 3 indicates that when the tumor DNAs were eluted from hydroxylapatite in 0.12 M phosphate buffer at 91°C, which was close to the TM for normal rat DNA (56.5% of the DNA melted), varied amounts of the native tumor DNAs were melted (44.6-73.0%). Although small changes in temperature ( < I°C) around the TM can Table 3. ii
seriously alter the amount of DNA melting, the results nonetheless suggest base compositional changes in the tumor DNAs. At any rate, the A + T rich fractions of BP, MC and DMBA tumor DNAs all showed significant reductions in their (dA) levels except for MC12 (P<0.10) (Table 3). However, the opposite was true for the G + C rich fractions where only 2 of the 8 tumor groups had
Evidencefor specificity of (dA ) alterations in the DNAs from rat tumors induced by polycyclic hydrocarbons.
N
Tumor inducing agent and identity number*
Hydroxylapatite thermal elution fractionation of DNA %• native DNA A + T rich DNA G + C rich DNA melting at 91°C % poly(U) hybrid t-test]" % poly(U) hybrid t-test]"
Normal rat tissue BP-10 BP-11 BP-15 MC-12 MC-13 MC-69 DMBA-14 .
i
__
56.5 44.6 62.3 67.3 55.0 60.5 73.0 50.1 I
ii
i
._
ii
0.136 _+0.019 0.107+0.019 0.121 ___0.018 0.095+0.022 0.125+0.023 0.104_+0.015 0.064_+ 0.017 0.114_+0.020 rl
illl
i
ii
i
-<0.0005 <0.025 <0.0005 <0.10 <0.0005 < 0.0005 <0.005
0.151 ___0.032 0.144___0.016 0.171 -t-0.041 0.096-t-0.017 0.146_+0.028 0.140+0.030 0.087 + 0.020 0.158_+0.024
-NS NS <0.0005 NS NS < 0.0005 NS
ii
The DNAs from groups of pooled tumors and from normal rat liver and embryo were loaded onto hydroxylapatite columns in 0.12M sodium phosphate buffer at 60°C and the A + T rich DNA fractions collected at 91°C (see text). The A + T and G + C fractions were immobilized onto nitrocellulose filters and annealed to [3H]-poly(U) for estimation of the (dA) regions (for further details see Ref. 7, 18 and text). *BP=3,4-ben~opyrene; MC=20-methylcholanthrene; DMBA= 7,12-dimethylbenz(a)anthracene. t P values for comparison to normal rat tissue; Means __+S.D. are shown; NS = not significant.
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Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and G6ran Levan
significant (dA) alterations (Table 3). These results were interpreted to mean that the majority of (dA) alterations specifically associated to neoplastic transformation induced by the polycyclic hydrocarbons must have occurred primarily in the A + T rich DNA. The possibility of base compositional changes in the polycyclic hydrocarbon induced tumor DNAs, which was previously suggested from our data in Table 3, was determined for 2, 2 and 4 groups of BP-, MC- and DMBAinduced tumors, respectively. The results in Table 4 indicate rather heterogeneous but statistically significant effects on the base composition of the tumors induced by the polycyclic hydrocarbons, where increases, decreases and' no changes at all were observed if comparison was made to normal rat DNA. However, all the groups of tumors examined in Table 4 regardless of whether their base compositions were increased, decreased or unchanged, still had significant reductions in their (dA) levels. As a result, a particular kind of overall base compositional change in native tun,or DNA was not a necessary prerequisite for demonstrating the (dA) disturbances associated with polycyclic hydrocarbon-induced carcinogenesis. Another striking parallel to our base analysis data is the fact that the DMBA-induced tumors tended to cause reductions in the % A + T in their native DNAs, whereas BP- and MC-induced tumors Table 4.
tended to cause increases or no changes at all in the o, A + T (Table 4). This study of (dA) alterations in neoplasm was carried out on the rat, but we have previously shown that similar neoplasticassociated (dA) disturbances can be found in the human [17, 18]. Therefore, we have compared the normal human and normal rat genomes to study if their (dA) regions are distributed within their respective genomic DNAs in a similar way. Table 5 demonstrates that (dA) regions can be found in essentially all classes of DNA from the rat and the human. However, the human differs in its (dA) distribution pattern from the rat by having a (dA) enrichment in main band human DNA when compared to the G + C rich and A + T rich human DNA fractions. The rat genome differed by having the same (dA) level in both G + C and main band rat DNA fractions. DISCUSSION
We have tried to show that rat tumors have small but detectable DNA template changes when compared to normal somatic cell rat DNA. Detection of these base changes have been carried out by a conventional base analysis method [26] and by utilization of a poly(U) probe to measure (dA) rich regions in polypurine rich DNA. In the latter case it
The base composition of DNAs from rat tumors induced by polycyclic hydrocarbons and its comparison to (dA ) alterations
Tumor inducing agent and identity number*
Base composition analysis n
% A+ T
t-test
% poly(U) annealing to DNA +
Normal rat tissue BP-10 MC-12 DMBA-14 BP-11 MC-13 DMBA- 17 DMBA-18 DMBA-19
38 12 10 13 13 18 11 15 5
58.6+ 1.8 59.7+2.2 58.6+1.6 58.4+2.3 60.0+2.4 60.6+ 1.6 56.7 + 1.9 55.6+2.0 54.9+0.1
-NS NS NS <0.05 <0.025 < 0.008 <0.0001 <0.0001
0.145+0.019 0.124+0.005§ 0.117+0.008§ 0.101 +0.006§ 0.107___0.005§ 0.104+0.008§ 0.134+0.008§ 0.098+0.010§ 0.112+0.011§
The DNAs from groups of pooled tumors and from normal rat liver and embryo were subjected to base composition analysis by measuring the reduction in u.v. absorption following bromination with N-bromoacetamide (for further details see Ref. 26 and the text). The same DNAs were also analyzed for (dA) alterations as reported in Table 1. *BP = 3,4-benzopyrene; MC = 20-methylcholanthrene; DMBA = 7,12-dimethylbenz(a)anthracene. t P values for comparison to normal rat tissues; N S = n o t significant; Means _ S.D. are shown. ++For estimation of (dA) regions divide % poly(U) value by 2. §P<0.025 (Student's t-test) when corrLpared to normal rat tissue; Means __+S.D. are shown.
Polycyclic Hydrocarbon-Induced Rat Sarcomas Table 5.
Source Rat Human
967
Genomic distribution of the (dA) regions in rat and human DNA fractional according to base composition in CsC1 gradients'. % poly(U) annealing to DNA of the pooled CsC1 fractions* G + C rich DNA Main band DNA A + T rich DNA 0.169__+0.008 0.128_+0.016
0.165__+0.011I" 0.162 _+0.012~
0.124___0.015 0.116_+0.016
Approximately 500-850#g of DNA from normal rat and human tissues were mixed with a CsCI solution to all initial density of 1,710 g/cm 3 and centrifuged for 72 hr at 44.000 rev/ndn in a Ti60 rotor. Twelve drop fractions were collected and then pooled into G + C rich (rat = 26.1%, human = 31.7%), main band (rat=33.3%, human=29.9%) and A + T rich (rat=40.6%, human=38.4%) fractions of the native DNAs. Each DNA fraction was then annealed to [3H]poly(U) for estimation of the (dA) regions as already described (see ReL 7 and tile text). *Means ___S.D. are shown. J'Statistically different (t-test, P<0.0005) from A + T rich DNA but not G + C rich DNA of the rat. +Statistically different (t-test, P<0.0005) from both G + C and A + T rich human DNAs.
can be argued that homopolymeric hybridizations to DNA have inherent methodological disadvantages, especially with formation of poly(U)-DNA hybrids which have low melting temperatures and are thermally more unstable. These disadvantages stem from the fact that the (dA) sequences are quite small in DNA and are close to the minimum stable length for molecular hybrid formation. As a result, we have shown that poly(U)-DNA hybrids are unusually sensitive to protein contamination, glycogen contamination, salt concentration, molecular weight, temperature and ribonuclease digestion [7, unpublished results]. However, we have been aware of these methodological difficulties and have taken them into consideration in our experiments. Nonetheless our results could have been influenced to some degree by such uncontrollable factors as specific tumorassociated nuclease effects on DNA preparation or acidic proteins bound very tightly and specifically to (dA) regions. Indeed these possibilities seem extremely remote explanations. Even if such explanations were proved to be responsible for our experimental observations, then our results would still indicate important biological variations of DNA that are associated to neoplasm. Our results on the (dA) disturbances in DNAs from polycyclic hydrocarbon-induced rat tumors have indicated that all were significantly altered from normal DNA (dA) levels. There was also quite a heterogeneous distribution in the degree of (dA) changes among the different chemically induced tumor groups and even within the tumor groups induced by the same carcinogen. Nevertheless, there was
a trend for the stronger carcinogens (DMBA and MC) to cause greater (dA) changes (Tables 1 and 2). This overall heterogeneity in distribution of (dA) changes among the various tumor groups can probably best be understood when considering the nature of chemically induced neoplasia. In such cases, there is an abnormally high exposure of carcinogen to target cells, and it is quite reasonable to suspect that high levels of DNA damage are inflected. Cells that do transform might carry not only base compositional changes associated with neoplasm but additional base changes not associated with neoplasm, and yet their presence in the genome does not alter cellular metabolism enough to cause a reduced cell survival. These non-neoplastic associated base changes would naturally vary from one tumor to another as a result of varying carcinogen reactivity in the original transforming cells. This type of base compositional change could involve (dA) regioffs, and if so would explain the observed heterogeneity in neoplastic associated (dA) changes. Other previous work [20, 21] has established that DMBA is the strongest carcinogen as it induces the highest frequency of tumors or cell transformations at the lowest doses. It has also been shown that DMBA binds more to DNA in cultured cells [29] and in epithelial homogenates [30] than does BP or MC. This is supportive evidence for the increased carcinogenic potential of DMBA when viewed from the mutational theory of cancer. Our data have also been able to distinguish the DNAs from DMBA-induced tumors from those of either BP- or MCinduced tumors. Although BP, MC and
968
Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and G6ran Levan
D M B A tumor DNAs all had significant reductions in the (dA) regions of their A + T rich D N A fractions when compared to similar normal D N A fractions (Table 3), only D M B A tumor DNAs were altered in base composition toward a more G + C rich D N A (an indication of preferential D M B A attack in A + T rich DNA, Table 4). Whether any correlation exists between the preference of (dA) alterations to appear in the A + T rich D N A fractions of tumors induced by the polycyclic hydrocarbons (Table 3) and the overall increased carcinogenicity of DMBA, still remains only speculative. However, if such a correlation were proved to be true it would be of importance to the mutational theory of cancer. We would like to point out that extreme caution should be exercised when extrapolating our results to neoplasms in animal systems other than the rat. Guttman et al. [31] have shown that the DNAs from 15 different mammals, analyzed by CsCl-netropsin density gradient centrifugation, contain quite different profiles with respect to their distribution patterns of (dA.dT) clusters. Indeed this has been our experience so far with normal and neoplastic DNAs from the rat and human. If we compared our TM-DNA fractionation experiments with human neoplastic DNAs [17] to
our similar experiments with rat tumor DNAs, then the (dA) regions in the A + T rich fractions of human neoplastic DNAs were increased over those in normal A + T rich human DNA, whereas in the rat tumor A + T rich DNAs the opposite was true (Table 3). O f course there are several reasonable explanations for this discrepancy such as the probable differences in oncogen between the human breast tumors and the rat tumors, or the fact that in the human experiments the T u fractionation at 88°C yielded a 46% fraction of A + T rich D N A and in the rat experiments the TM fractionation was carried out at 91°C and yielded a 56.5% fraction of A + T rich DNA. However, still the most likely explanation for the varying neoplastic-associated disturbances observed between the rat and human, is probably correlated to differences in genomic distribution of (dA) regions. The data in Table 5 supports this argument. The (dA) regions in normal rat DNA are distributed equally into the G + C rich and main band DNAs but in the normal human genome they are not.
Acknowledgements--The
authors are indebted to Dr. Felix Mitelman for supply of transplanted DMBA tumors and to Dr. Albert Levan for his contributions to the genetic evaluation of some of the rat sarcomas.
REFERENCES 1. H. C. BIRNBOIM,R. E. J. MITCHEL and N. A. STRAUS, Analysis of long pyrimidine polynucleotides in Hela cell nuclear DNA: absence of polydeoxythymidylate. Proc. nat. Acad. Sci. (Wash.)70, 2189 (1973). 2. H. C. BIRNBOIM and N. A. STRAUS, DNA from eukaryotic cells contains unusually long pyrimidine sequences. Canad. 07. Biochem. 53, 640 (1975). 3. J. O. BISHOP, M. ROSBASH and D. EVANS, Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid. 07. mol. Biol. 85, 75 (1974). 4. S.T. CASE and R. F. BAKER, Detection of long eukaryotic specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos. 07. tool. Biol. 98, 69 (1975). 5. H. KUBINSKI, OPARA-KuBINSKA and W. SZYBALSKI, Patterns of interaction between polyribonucleotides and individual DNA strands derived from several vertebrates, bacteria and bacteriophages. 07. tool. Biol. 20, 313 (1966). 6. J. N. M. MOL and P. BORST, The binding of poly(rA) and poly(rU) to denatured DNA. II. Studies with natural DNAs. Nucleic Acids Res. 3, 1029 (1976). 7. R . W . PERO, T. BRYNGELSSON, C. BRYNGELSSON, A. DEUTSCH, A. NORDf~N and A. NORGREN, Hybridization of polyuridylic acid to human DNA immobilized onto nitrocellulose filters. Nucleic Acids Res. 2, 1163 (1975). 8. R. SEDEROFF, L. LOWENSTEINand H. C. BIRNBOIM,Polypyrimidine segments in Drosophila melanogaster DNA. II. Chromosome location and nucleotide sequence. Cell 5, 183 (1975). 9. A. SHENKIN and R. H. BORDON, Deoxyadenylate-rich sequences in mammalian DNA. FEBS Lett. 22, 157 (1972). 10. A. SHENKIN and R. N. BUROON,Deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA. 07. mol. Biol. 85, 19 (1974).
Polycyclic Hydrocarbon-Induced Rat Sarcomas 11. 12. 13.
14.
15. 16. 17.
18.
19.
20.
21.
22. 23. 24. 25. 26.
27.
28. 29.
30.
31.
N. A. STRAUS and H. C. BIRNBOIM,Long pyrimidine tracts of L-cell DNA: localization to repeated DNA. Proc. nat. Acad. Sci. (Wash.) 71, 2992 (1974). R. H. BURDON and A. SHENKIN,Uridylate-rich sequences in rapidly labelled RNA of mammalian cells. P~BS Lett. 24, 11 (1972). G . R . MOLLOY, W. L. THOMAS and J. E. DARNELL, Occurrence of uridylaterich oligonucleotide regions in heterogenous nuclear RNA of HeLa cells. Proc. nat. Acad. Sci. (Wash.) 69, 3684 (1972). H. NAKAZATO, M. EDMUNDS and D. W. KoPP, Differential metabolism of large and small poly(A) sequences in heterogeneous nuclear RNA of Hela cells. Proc. nat. Acad. Sci (Wash.) 71, 200 (1974). B. LEWlN, Units of transcription and translation: Sequence components of heterogeneous nuclear RNA and messenger RNA. Cell 4, 77 (1975). B. LEWIN, Units of transcription and translation, the relationship between heterogeneous nuclear RNA and messenger RNA. Cell 4, 11 (1975). R . W . PERO, T. BRYNGELSSON,A. NORGREN and A. DEUTSCH,Alterations in the deoxyadenylate regions of the DNA from four human breast tumors. Europ. J. Cancer 11, 861 (1975). R . W . PERO, T. BRYNGELSSON,A. DEUTSCH and ,~. NORD~N, Changes in the deoxyadenylate regions of DNA in 5 cases of chronic lymphocytic leukemia. Europ. J. Cancer 12, 357 (1976). R . W . PERO, T. BRVNOELSSON, F. MITELMAN and G. LEVAN, Changes in the deoxyadenylate regions of rat DNA in sarcomas induced by 7,12dimethylbenz (a )anthracene and Rous sarcoma virus. Hereditas 80, 153 (1975). T. T. CHEN and C. HEXBELBERGER, Quantitative studies on the malignant transformation of mouse prostate cells by carcinogenic hydrocarbons in vitro. Int. J. Cancer 4, 166 (1969). E. D. REES, Chromosomal aberrations in rat marrow cells following intravenous polycyclic hydrocarbons: possible basis for a carcinogen bioassay. In Proceedings in the Tobacco Health Workshop. Vol. 18, p. 42. University of Kentucky Tobacco Health Research Institute, Lexington (1969). G. LLwtx, U. AHLSTR6M and F. MITELMAN,The specificity of chromosome .\2 iuvohement in DMBA-induced rat sarcomas. Hereditas 77, 263 (1974). F. MITELMAN, Predetermined sequential chromosome changes in serial transplantation of Rous rat sarcomas. Acta path. microbiol, scand. 80, 313 (1972). J. MARMUR, A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. mol. Biol. 3, 208 (1961). J. WmcocKsoy, The use of sodium perchlorate in deproteinization during the preparation of nucleic acids. Biochem. J. 135, 559 (1973). S.Y. WAN% The determination of nucleic acid base composition by chemical reactivity. In Methods in Enzymology. (Edited by L. Grossman and K. Moldave) Vol. 12, part B, p. 178. Academic Press, New York (1968). G. LEVAN and A. LEVAN,Specific chromosome changes in malignancy: studies in rat sarcomas induced by two polycyclic hydrocarbons. Hereditas 79, 161 (1975). F. MITELMAN and G. LEVAN, The chromosomes of primary 7,12dimethylbenz(a)anthracene-induced rat sarcomas. Hereditas 71~ 325 (1971). M. DUNCAN,P. BROOKESand A. DIPPLE, Metabolism and binding to cellular macromolecules of a series of hydrocarbons by mouse embryo cells in culture. Int. J. Cancer 4, 813 (1969). T . J . SLAGA, S. G. BUTY, S. THOMPSON,W. M. BRACKENand A. VIAJE, A kinetic study on the in vitro covalent binding of polycyclic hydrocarbons to nucleic acids using epidermal homogenates as the activating system. Cancer Res. 37, 3126 (1977). T. GUTTMAN, H. VOTOVOV£ and L. PIVEC, Base composition heterogeneity of mammalian DNAs in CsCl-netropsin density gradient. Nucleic Acids Res. 3, 835 (1976).
969
0111 )-291i~ 78, 0901-0961 S02.00,'0
Polycyclic Hydrocarbon-Induced Rat Sarcomas Correlated to Disturbances in the Deoxyadenylate Regions of the Tumor DNAs* RONALD W. PERO,~ TOMAS BRYNGELSSON,+ CHRISTINA RUDDUCK+ and GORAN LEVAN++ t Nucleic Acid Biochemistry and +Institute of Genetics, Lund University, Wallenberg Laboratory, Fack 7031,220 07 Lund, Sweden A b s t r a c t - - T e n separate groups containing a total of 51 rat sarcomas induced by the polycyclic hydrocarbons were examined for inducing agent-related abnormalities in their DNAs. The levels of deoxyadenylate (dA) regions in normal and tumor rat DNAs were estimated from annealing [3H]-polyuridylic acid to D N A and treatment of the resultant hybrids with ribonuclease A. All the tumor material had significant reductions in the (dA ) regions of their DNAs when the levels were compared to normal rat DNA. Although the (dA) reductions were rather heterogeneous from one tumor group to the other no matter the inducing agent, the greatest decreases tended to be found with tumors induced by 20-methylcholanthr ene (M C ) and 7,12-dimethylbenz(a)anthracene (DMBA). No relationship was found between (dA) alterations and the types of chromosomal aberrations in the tumor cell populations. However, normal and tumor DNAs, fractionated into A + T rich and G + C rich D N A by thermal elution from hydroxylapatite columns, showed specific neoplastic-associated (dA ) disturbances in the A + T rich D N A of tumors induced by 3,4-benzopyrene (BP), M C and D M B A . The base compositions of the tumor material were also analyzed and compared to normal rat D.\:t. Tumors induced by D M B A showed highly significant decreases in ~o A + T ~'hereas B P and M C induced tumors tended to show only small increases in o i) A + T. Some tumors induced by BP, M C and D M B A were unaltered in base composition. Together our results are consistent with the hypothesis that D M B A is the strongest carcinogen in the series because it interacts preferentially with A + T rich DNA, which is the type of D N A most important for polycyclic hydrocarbon-induced neoplasm in the rat.
INTRODUCTION
other polypyrimidine stretches were approximately 25-200 nucleotides in length [2-4, 6, 10] and were concentrated into repeated sequences in the D N A [4, 7, 11]. The fact that these oligohomopolymeric rich regions could not have arisen from a random selection model [1], together with their specificity for location into repeated DNA, have strongly suggested a biological function for these regions. These observations are further supported from similar work on nRNA. It has been shown that oligo(U) and oligo(A) regions of approximately 25-35 nucleotides are a normal component of n R N A [12-14] indicating that their template for transcription must have been the (dA) or (dT) i'egions in the DNA [13]. The oligo (U) regions in n R N A have also been shown to be located primarily in the
THE PRESENCE of (dA) rich stretches and polypyrimidine runs in eukaryotic D N A has now been validated by several laboratories [1-11]. The (dA) or (dT) rich sequences and
Accepted 12 January 1978. *This work was supported by the Swedish Cancer Society (RMC 76:101) and by a special grant to the Dalby Community Care Sciences program from the National Board of Health and Social Welfare in Sweden. The abbreviations used are: (dA), deoxyadenylate; (dT), deoxythymidylate; (U), ribouridylate; (A), riboadenylate; BP, 3,4-benzopyrene; MC, 20-methyleholanthrene, DMBA, 7,12-dimethylbenz(a)anthracene; SSC, 0.15M NaCI+0.015M sodium citrate, pH=7.2; Txl, temperature at which 50% native DNA becomes single stranded nRNA, nuclear RNA. 961
9~
Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and G6ran Levan and not karyotyped. The groups of tumors designated DMBA-17 and DMBA-19 were transplanted for a single passage in newborn Wistar-Furth rats as described elsewhere [23]. All tumors were frozen and stored at - 7 0 ° C until used for DNA extraction. The livers, spleens, inner skin covering the abdominal cavity and full term embryos were dissected from adult Wistar-Furth rats and either processed directly or stored at - 7 0 ° C . These tissues were taken from apparently healthy animals and served as the control sources of normal rat DNA.
5'-end of n R N A which is mainly transcribed from repeated DNA template [14, 15] and confirms the location of (dA) clusters there. Since the 3'-end of n R N A contains unique DNA transcript or the mRNA, then the implications are that (dA) transcripts or the oligo(U) regions might play ~ role in the proposed processing of n R N A into m R N A [16]. At any rate, the exact function of (dA) transcripts is still unknown although some biological function most certainly can be implied. Our laboratory has assumed an important function for the (dA) regions in either the transcription or processing of genetic information, and has attempted to assign a role to them in neoplasm. Our results have shown that (dA) alterations could be demonstrated in the DNA from human breast tumors [17], human CLL lymphocytes [18] and rat tumors induced by Rous sarcoma virus or DMBA [19]. Generally, the more malignant cell types were associated with reduced levels of (dA) regions in their DNAs. Although (dA) disturbances were found in both A + T rich and G + C rich DNA [17], some specificity of change was observed for both main band and low repeated DNAs (18). However, our ability to demonstrate specific (dA) alterations common to neoplasm has been complicated by looking at neoplasms where the oncogenic agent is not known. Presumably, the (dA) alterations caused from viral carcinogenesis where the viral genome is involved could be quite different from those arising from chemical carcinogens. Therefore, in this study we have examined rat tumors induced by a class of closely related carcinogens, the polycyclic hydrocarbons, and where the carcinogenic potency varies in a predicted way [20, 21]. With this well defined tumor induction model, we have tried to show that tumors induced by structurally-related chemicals have (dA) alterations in specific areas of their DNA.
Extraction of DNA The rat tissues were minced in ice cold 0.1M E D T A - 0 . 1 M NaCI, pH 7.5 and homogenized at 4°C by 2 x 15-sec pulses at 1 n~lin intervals with an Ultra-Turrax. The DNA was extracted from the homogenates by the M a r m u r method [24] except for the tinal alcohol precipitation. Instead of the precipitation step, 100 #g/ml ribonuclease A (Sigma) and 100#g/ml alpha amylase (Sigma) were added to the DNA solutions and the mixture dialyzed overnight against 51 0.1 x SSC at room temperature. Final!y, the enzyme-treated DNA solutions were made 5% sodium dodecyl sulfate and precipitated with alcoholic sodium perchlorate solution as described by Wilcockson [25]. In our hands, DNA prepared in this way was essentially free from glycogen (anthrone method), RNA (orcinol method) and protein of which contamination was always less than 5% (#g protein/pg DNA, Lowry method). We have noticed in our liver preparations from unstarved animals that high concentrations of glycogen (100 #g/ml DNA solution) can occur in our final DNA preparations, if alpha amylase treatment was omitted. We have also observed that such high levels of glycogen contamination of DNA interferes with the poly(U) hybridization on filters (data not shown).
MATERIALS AND M E T H O D S Tumor induction and normal tissue Inbred Wistar-Furth rats 8-35 days of age were inoculated subcutaneously in the right thigh with 1 - 4 m g doses of BP, MC or D~IBA dissolved in 0.1 ml tricaprylin. T u m o r s developed to 100% and were harvested 6-8 weeks after they were palpable ( < 7 months). Some individual tumors were analyzed for karyotypic abnormalities by direct fixation according to Levan et al. [22]. Other tumors induced by the same carcinogen were pooled
Poly( U) annealing method We have described elsewhere [7] the essential details of our poly(U) hybridization method and the factors which limit its reproducibility. However, we have now introduced modifications which have reduced much of the variability encountered in our earlier experiments. Approximately 15#g samples of DNA dissolved in 2 x SSC were denatured by heating at 100°C for 10 rain followed by another 10 min with an equal volume of 4 M NaCI. Treating denatured DNA with hot 2 M NaCI insured retention of (dA) rich sequences
Polycyclic Hydrocarbon-Induced Rat Sarcomas and improved on selective removal of any residual contaminating protein [7]. The solutions were cooled immediately in an ice bath, loaded onto nitrocellulose filters (Millipore ®) with suction and baked in an oven for 2--4hr at 80°C. In order to improve on our yield of DNA from smaller tumors, the alcohol precipitated DNA was often collected by centrifugation. These DNA preparations tended to contain lower molecular weight DNA fragments that are normally left behind when the DNA is spooled and thus these DNA solutions retained less on the filters. Previously severely sheared DNA affected the annealing of poly(U) [7]. However, we have compared only DNAs which retained between 75-100% on the filters, and with this level of retention the estimation of (dA) regions was unaffected by the amount of DNA immobilized (data not shown). Commercial [3H]poly(U) (Miles Laboratory, 75pCi//~M monophosphate) was dissolved in 2 x SSC and adjusted to a final concentration of 6.0#g/ml with unlabelled poly(U) (Sigma). The DNA containing filters were placed in the [3H]poly(U) solution and annealed for 17hr at 25°C with continuous mixing from the aid of a magnetic stirrer. Unannealed poly(U) was removed from the filters by ribonuclease A treatment in 2 x SSC at 5 #g/ml for 6 hr at 25°C. It was important, especially in cases where many filters were treated batchwise, to have efficient stirring for both the annealing and RNAase steps if unnecessary variability was to be avoided. The details involving radioactive counting on filters, quantitative determination of DNA and estimation of (dA) "pure" sequences from the °/o poly(U) annealing to DNA after 6 hr RNAase treatment were unchanged from our earlier studies [7]. Routine poly(U) analyses were replicated 1030 times. The amount of DNA hybridizing to poly(U) was always determined on the nitrocellulose filters after RNAase treatment and scintillation counting.
TMfractionation of DNA on hydroxylapatite The TM fractionation was carried out as we have described earlier for human DNA [17] except for a few minor details. Native DNA was loaded onto hydroxylapatite in 0.01 M phosphate buffer at 60°C and the A + T rich DNA fraction eluted between 60 and 91°C in 0.12 M phosphate buffer.
Base composition analysis The method used for base analysis was the same as reported by Wang [26]. Briefly, it is
based on the fact that all the bases except adenine react with N-bromoacetamide to cause reduction in U V absorption. Standard solutions of dAMP, dCMP, d G M P and d T M P were prepared in 0.5 M H2SO4. Since at this acid concentration d A M P and d G M P were depurinated, then the molar absorbancy coefficients used to adjust the final standard concentrations to 0.05raM were as given: adenine ( E 2 7 0 = l l . 5 6 x 1 0 - 3 ) , d C M P (E270 = 1 1 . 4 4 x 1 0 - 3 ) , guanine ( E 2 v 0 = 7 . 4 x 1 0 .3 ) and d T M P (E27o=9.58 x 10-3). Mixtures of the 4 standard solutions were used to prepare a standard curve ranging from 100% A + T to 100% G + C . It was necessary in our experiments to heat the DNA solutions in 0.5M H2SO 4 for 3hr at 50°C to insure complete depurination and allow accurate estimation of base composition. Other important details such as the preparation and addition of the brominating agent and the calculation of % A + T from the u.v. absorption changes were carried out as described by Wang [26]. RESULTS
A total of 51 rat sarcomas induced by the polycyclic hydrocarbons were pooled into 10 different groups and compared to normal tissues for alterations in the (dA) regions of their DNAs. A quantitative estimation of the (dA) regions in the DNA from normal rat tissue was always the same whether the sources used were embryo, skin or liver + spleen (Table 1). A constant level of (dA) regions in normal somatic cell DNA of the rat has thus provided the analytical basis for its comparison to the (dA) levels in neoplastic DNA. All groups of tumors reported in Table 1 regardless of inducing agent had statistically significant reductions in the (dA) regions of their DNAs. The level of (dA) reductions in the tumor DNAS varied considerably from one tumor group (MC-69) to another (DMBA17), but there was a tendency for MC- and DMBA-induced tumors to have the greatest (dA) reductions (Table 1). Such heterogeneity in the (dA) alterations of DNA from tumors induced with so structurally similar carcinogens, or even when induced by the same carcinogen (i.e., MC-12 and MC-69), suggests that additional DNA template changes involving in part the (dA) regions, have accompanied those DNA changes responsible for the neoplastic event itself. One possible way to judge the degree of additional DNA changes which might occur along with those DNA changes necessary for
964
Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and Go'ran Levan Table 1. Comparison of normal tissues to polycyclic hydrocarbon-induced tumors in the rat by quantitative analysis of the (dA ) regions in their DNAs. Tumor inducing agent and identity number* Normal rat tissues embryo skin liver and spleen BP-10 BP-I 1 BP-15 MC-12 MC- 13 MC-69 DMBA- 17 DMBA-14 DMBA- 18 DMBA-19
Number of pooled tumors
% poly(U) annealing to D N A J "
Average %/tumor/agent++
---4 5 5 6 7 4 5. 6 3 6
0.147 4- 0.017 0.145 -t-0.015 0.142 ___0.023 0.12t _ 0.005§ 0,107 + 0.005§ 0.121 _ 0.019§ 0.117 4-0.008§ 0.104 4- 0.008§ 0.059 4- 0.009§ 0.134 4- 0.008§ 0.101 4-0.006§ 0.112 4-0.011§ 0.098 4-0.010§
--0.145 --0.I 17 --0.098 ---0.110
The DNAs from groups of pooled tumors and from normal tissues of the rat were extracted, immobilized onto nitrocellulose filters and annealed to [aH]poly(U) in 2 x SSC at 25°C for 18hr. Non-specific binding of [3H]-poly(U) was removed by treatment of the filters with 5/~g/ml ribonuclease A dissolved in 2 x SSC for 6 hr (for further details see Ref. 7 and the text). *BP = 3,4-benzopyrene; MC = 20-methylcholanthrene; DMBA = 7,12-dimethylbenz (a)anthracene. J'For estimation of (dA) regions divide % poly(U) value by 2. ~.%/tumor/agent represents the average % poly(U) annealing to the DNA from all the tumors induced by the same polycyclic hydrocarbon. Hence, the average (dA) alterations expected for a tumor induced by particular polycyclic hydrocarbon in question. §P<0.0005 (Student's t-test) when compared to the values ~br normal rat tissues; DMBA-14 (p<0.025); Means +S.D. are shown. polycyclic hydrocarbon-induced neoplastic t r a n s f o r m a t i o n , m i g h t be to c o m p a r e those t u m o r s w h i c h contain high frequencies of either similar or different c h r o m o s o m a l a b e r rations. I t has b e e n s h o w n [27, 28] t h a t specific c h r o m o s o m a l a b n o r m a l i t i e s are associated with t u m o r s i n d u c e d b y the polycyclic h y d r o c a r b o n s , a n d some o f these a b n o r m a lities such as the trisomy A2 can be found in t u m o r s i n d u c e d b y all 3 o f the carcinogens BP, M C a n d D M B A [22, 27]. I n T a b l e 2 we h a v e selected BP- a n d M C - i n d u c e d t u m o r s which h a d high incidences of a p a r t i c u l a r type o f c h r o m o s o m a l a b e r r a t i o n in the t u m o r cell populations. T h i s protocol has allowed us to try a n d s t a n d a r d i z e the initial a m o u n t of D N A d a m a g e inflected b y the c a r c i n o g e n at the t i m e o f t r a n s f o r m a t i o n , as evidenced by the similar c h r o m o s o m a l a b e r r a t i o n s in the s u b s e q u e n t t u m o r cell p o p u l a t i o n , a n d to test w h e t h e r these c h r o m o s o m a l similarities w e r e specific e n o u g h to reflect similar (dA) alterations. T a b l e 2 clearly d e m o n s t r a t e s t h a t a high incidence of a distinct c h r o m o s o m a l deviation such as trisomy or the presence of a m a r k e r c h r o m o s o m e in the t u m o r cell p o p u -
lation, or even the absence of a b n o r m a l i t i e s altogether, does not predict the neoplasticassociated (dA) alterations in the D N A . A p p a r e n t l y the presence of other r a n d o m c h r o m o s o m a l a b e r r a t i o n s in the t u m o r cells m u s t m a s k a n y specificity shown b y the n o n r a n d o m , distinct c h r o m o s o m a l deviations, at least, as far as a n y correlation to (dA) alterations are concerned. I n fact, s u p p o r t for this i n t e r p r e t a t i o n c a n be d r a w n f r o m the fact, as we a l r e a d y observed in T a b l e 1, t h a t again there was a r a t h e r good correlation to inducing agent; n a m e l y , M C - i n d u c e d tumors all h a d g r e a t e r reductions in the (dA) regions of their D N A s t h a n did B P - i n d u c e d t u m o r s ( T a b l e 2). Such a correlation to i n d u c i n g a g e n t would take into a c c o u n t the total altered g e n o m e a n d not just the c h r o m o s o m a l a b e r r a t i o n s w h i c h were either specific, nonspecific or absent. W e h a v e further p u r s u e d o u r efforts to distinguish the m o r e specific neoplasticassociated (dA) changes f r o m those nonessential D N A changes which m i g h t a c c o m p a n y polycyclic h y d r o c a r b o n induction of rat sarcomas, b y m e a s u r i n g the (dA) levels in A
,%5
Polycyclic Hydrocarbon-Induced Rat Sarcomas Table 2. Lack of correlation between the chromosomal aberrations in polycyclic hydrocarbon-induced rat tumors and the (dA) alterations of their DNAs Tumor inducing agent and identity number*
Chromosomal aberration specificity
% poly(U) annealing to DNA]'
Normal rat tissue BP- 1 BP-2 MC-6 MC-7 BP-3 MC-8 BP-4 MC-9 BP-5
normal normal trisomy A2 trisomy A2 trisomy A2 marker i(2) marker i(2) marker t(3;?) marker t(3;?) marker t(11;?)
0.145 _+0.019 0.101 _+0.017 + 0.134_+ 0.017++ 0.055 _+0.002+ 0.056 _+0.003++ 0.134_+0.011++ 0.070 -t-0.002++ 0.118 +0.017++ 0.054-t-0.017++ 0.111 -+0.018++
The DNAs from individual rat tumors which had been karyotyped for any specific chromosomal abnormalities and from normal rat skin and liver were extracted, immobilized onto nitrocellulose filters and annealed to [3H]-poly(U) for estimation of (dA) clusters (for further details see Ref. 7 or text). Additional details of the chromosomal aberration designations are found in Ref. 27. *BP=3,4-benzopyrene; MC=20methylcholanthrene; The identity numbers in this table correspond to the following identification numbers in Ref. 27 presented in order from top of table to bottom: BP10, BP12, MC8, MC1, BP9, MC2, BP2, MC2+5, BP4. ]'For estimation of (dA) regions divide % poly(U) value by 2. ,*P<0.05 (Student's t-test) when corripared to value for normal rat tissue; Means _+S.D. are shown.
+ T rich and G + C rich subfractions of the native tumor DNAs. Table 3 indicates that when the tumor DNAs were eluted from hydroxylapatite in 0.12 M phosphate buffer at 91°C, which was close to the TM for normal rat DNA (56.5% of the DNA melted), varied amounts of the native tumor DNAs were melted (44.6-73.0%). Although small changes in temperature ( < I°C) around the TM can Table 3. ii
seriously alter the amount of DNA melting, the results nonetheless suggest base compositional changes in the tumor DNAs. At any rate, the A + T rich fractions of BP, MC and DMBA tumor DNAs all showed significant reductions in their (dA) levels except for MC12 (P<0.10) (Table 3). However, the opposite was true for the G + C rich fractions where only 2 of the 8 tumor groups had
Evidencefor specificity of (dA ) alterations in the DNAs from rat tumors induced by polycyclic hydrocarbons.
N
Tumor inducing agent and identity number*
Hydroxylapatite thermal elution fractionation of DNA %• native DNA A + T rich DNA G + C rich DNA melting at 91°C % poly(U) hybrid t-test]" % poly(U) hybrid t-test]"
Normal rat tissue BP-10 BP-11 BP-15 MC-12 MC-13 MC-69 DMBA-14 .
i
__
56.5 44.6 62.3 67.3 55.0 60.5 73.0 50.1 I
ii
i
._
ii
0.136 _+0.019 0.107+0.019 0.121 ___0.018 0.095+0.022 0.125+0.023 0.104_+0.015 0.064_+ 0.017 0.114_+0.020 rl
illl
i
ii
i
-<0.0005 <0.025 <0.0005 <0.10 <0.0005 < 0.0005 <0.005
0.151 ___0.032 0.144___0.016 0.171 -t-0.041 0.096-t-0.017 0.146_+0.028 0.140+0.030 0.087 + 0.020 0.158_+0.024
-NS NS <0.0005 NS NS < 0.0005 NS
ii
The DNAs from groups of pooled tumors and from normal rat liver and embryo were loaded onto hydroxylapatite columns in 0.12M sodium phosphate buffer at 60°C and the A + T rich DNA fractions collected at 91°C (see text). The A + T and G + C fractions were immobilized onto nitrocellulose filters and annealed to [3H]-poly(U) for estimation of the (dA) regions (for further details see Ref. 7, 18 and text). *BP=3,4-ben~opyrene; MC=20-methylcholanthrene; DMBA= 7,12-dimethylbenz(a)anthracene. t P values for comparison to normal rat tissue; Means __+S.D. are shown; NS = not significant.
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Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and G6ran Levan
significant (dA) alterations (Table 3). These results were interpreted to mean that the majority of (dA) alterations specifically associated to neoplastic transformation induced by the polycyclic hydrocarbons must have occurred primarily in the A + T rich DNA. The possibility of base compositional changes in the polycyclic hydrocarbon induced tumor DNAs, which was previously suggested from our data in Table 3, was determined for 2, 2 and 4 groups of BP-, MC- and DMBAinduced tumors, respectively. The results in Table 4 indicate rather heterogeneous but statistically significant effects on the base composition of the tumors induced by the polycyclic hydrocarbons, where increases, decreases and' no changes at all were observed if comparison was made to normal rat DNA. However, all the groups of tumors examined in Table 4 regardless of whether their base compositions were increased, decreased or unchanged, still had significant reductions in their (dA) levels. As a result, a particular kind of overall base compositional change in native tun,or DNA was not a necessary prerequisite for demonstrating the (dA) disturbances associated with polycyclic hydrocarbon-induced carcinogenesis. Another striking parallel to our base analysis data is the fact that the DMBA-induced tumors tended to cause reductions in the % A + T in their native DNAs, whereas BP- and MC-induced tumors Table 4.
tended to cause increases or no changes at all in the o, A + T (Table 4). This study of (dA) alterations in neoplasm was carried out on the rat, but we have previously shown that similar neoplasticassociated (dA) disturbances can be found in the human [17, 18]. Therefore, we have compared the normal human and normal rat genomes to study if their (dA) regions are distributed within their respective genomic DNAs in a similar way. Table 5 demonstrates that (dA) regions can be found in essentially all classes of DNA from the rat and the human. However, the human differs in its (dA) distribution pattern from the rat by having a (dA) enrichment in main band human DNA when compared to the G + C rich and A + T rich human DNA fractions. The rat genome differed by having the same (dA) level in both G + C and main band rat DNA fractions. DISCUSSION
We have tried to show that rat tumors have small but detectable DNA template changes when compared to normal somatic cell rat DNA. Detection of these base changes have been carried out by a conventional base analysis method [26] and by utilization of a poly(U) probe to measure (dA) rich regions in polypurine rich DNA. In the latter case it
The base composition of DNAs from rat tumors induced by polycyclic hydrocarbons and its comparison to (dA ) alterations
Tumor inducing agent and identity number*
Base composition analysis n
% A+ T
t-test
% poly(U) annealing to DNA +
Normal rat tissue BP-10 MC-12 DMBA-14 BP-11 MC-13 DMBA- 17 DMBA-18 DMBA-19
38 12 10 13 13 18 11 15 5
58.6+ 1.8 59.7+2.2 58.6+1.6 58.4+2.3 60.0+2.4 60.6+ 1.6 56.7 + 1.9 55.6+2.0 54.9+0.1
-NS NS NS <0.05 <0.025 < 0.008 <0.0001 <0.0001
0.145+0.019 0.124+0.005§ 0.117+0.008§ 0.101 +0.006§ 0.107___0.005§ 0.104+0.008§ 0.134+0.008§ 0.098+0.010§ 0.112+0.011§
The DNAs from groups of pooled tumors and from normal rat liver and embryo were subjected to base composition analysis by measuring the reduction in u.v. absorption following bromination with N-bromoacetamide (for further details see Ref. 26 and the text). The same DNAs were also analyzed for (dA) alterations as reported in Table 1. *BP = 3,4-benzopyrene; MC = 20-methylcholanthrene; DMBA = 7,12-dimethylbenz(a)anthracene. t P values for comparison to normal rat tissues; N S = n o t significant; Means _ S.D. are shown. ++For estimation of (dA) regions divide % poly(U) value by 2. §P<0.025 (Student's t-test) when corrLpared to normal rat tissue; Means __+S.D. are shown.
Polycyclic Hydrocarbon-Induced Rat Sarcomas Table 5.
Source Rat Human
967
Genomic distribution of the (dA) regions in rat and human DNA fractional according to base composition in CsC1 gradients'. % poly(U) annealing to DNA of the pooled CsC1 fractions* G + C rich DNA Main band DNA A + T rich DNA 0.169__+0.008 0.128_+0.016
0.165__+0.011I" 0.162 _+0.012~
0.124___0.015 0.116_+0.016
Approximately 500-850#g of DNA from normal rat and human tissues were mixed with a CsCI solution to all initial density of 1,710 g/cm 3 and centrifuged for 72 hr at 44.000 rev/ndn in a Ti60 rotor. Twelve drop fractions were collected and then pooled into G + C rich (rat = 26.1%, human = 31.7%), main band (rat=33.3%, human=29.9%) and A + T rich (rat=40.6%, human=38.4%) fractions of the native DNAs. Each DNA fraction was then annealed to [3H]poly(U) for estimation of the (dA) regions as already described (see ReL 7 and tile text). *Means ___S.D. are shown. J'Statistically different (t-test, P<0.0005) from A + T rich DNA but not G + C rich DNA of the rat. +Statistically different (t-test, P<0.0005) from both G + C and A + T rich human DNAs.
can be argued that homopolymeric hybridizations to DNA have inherent methodological disadvantages, especially with formation of poly(U)-DNA hybrids which have low melting temperatures and are thermally more unstable. These disadvantages stem from the fact that the (dA) sequences are quite small in DNA and are close to the minimum stable length for molecular hybrid formation. As a result, we have shown that poly(U)-DNA hybrids are unusually sensitive to protein contamination, glycogen contamination, salt concentration, molecular weight, temperature and ribonuclease digestion [7, unpublished results]. However, we have been aware of these methodological difficulties and have taken them into consideration in our experiments. Nonetheless our results could have been influenced to some degree by such uncontrollable factors as specific tumorassociated nuclease effects on DNA preparation or acidic proteins bound very tightly and specifically to (dA) regions. Indeed these possibilities seem extremely remote explanations. Even if such explanations were proved to be responsible for our experimental observations, then our results would still indicate important biological variations of DNA that are associated to neoplasm. Our results on the (dA) disturbances in DNAs from polycyclic hydrocarbon-induced rat tumors have indicated that all were significantly altered from normal DNA (dA) levels. There was also quite a heterogeneous distribution in the degree of (dA) changes among the different chemically induced tumor groups and even within the tumor groups induced by the same carcinogen. Nevertheless, there was
a trend for the stronger carcinogens (DMBA and MC) to cause greater (dA) changes (Tables 1 and 2). This overall heterogeneity in distribution of (dA) changes among the various tumor groups can probably best be understood when considering the nature of chemically induced neoplasia. In such cases, there is an abnormally high exposure of carcinogen to target cells, and it is quite reasonable to suspect that high levels of DNA damage are inflected. Cells that do transform might carry not only base compositional changes associated with neoplasm but additional base changes not associated with neoplasm, and yet their presence in the genome does not alter cellular metabolism enough to cause a reduced cell survival. These non-neoplastic associated base changes would naturally vary from one tumor to another as a result of varying carcinogen reactivity in the original transforming cells. This type of base compositional change could involve (dA) regioffs, and if so would explain the observed heterogeneity in neoplastic associated (dA) changes. Other previous work [20, 21] has established that DMBA is the strongest carcinogen as it induces the highest frequency of tumors or cell transformations at the lowest doses. It has also been shown that DMBA binds more to DNA in cultured cells [29] and in epithelial homogenates [30] than does BP or MC. This is supportive evidence for the increased carcinogenic potential of DMBA when viewed from the mutational theory of cancer. Our data have also been able to distinguish the DNAs from DMBA-induced tumors from those of either BP- or MCinduced tumors. Although BP, MC and
968
Ronald W. Pero, Tomas Bryngelsson, Christina Rudduck and G6ran Levan
D M B A tumor DNAs all had significant reductions in the (dA) regions of their A + T rich D N A fractions when compared to similar normal D N A fractions (Table 3), only D M B A tumor DNAs were altered in base composition toward a more G + C rich D N A (an indication of preferential D M B A attack in A + T rich DNA, Table 4). Whether any correlation exists between the preference of (dA) alterations to appear in the A + T rich D N A fractions of tumors induced by the polycyclic hydrocarbons (Table 3) and the overall increased carcinogenicity of DMBA, still remains only speculative. However, if such a correlation were proved to be true it would be of importance to the mutational theory of cancer. We would like to point out that extreme caution should be exercised when extrapolating our results to neoplasms in animal systems other than the rat. Guttman et al. [31] have shown that the DNAs from 15 different mammals, analyzed by CsCl-netropsin density gradient centrifugation, contain quite different profiles with respect to their distribution patterns of (dA.dT) clusters. Indeed this has been our experience so far with normal and neoplastic DNAs from the rat and human. If we compared our TM-DNA fractionation experiments with human neoplastic DNAs [17] to
our similar experiments with rat tumor DNAs, then the (dA) regions in the A + T rich fractions of human neoplastic DNAs were increased over those in normal A + T rich human DNA, whereas in the rat tumor A + T rich DNAs the opposite was true (Table 3). O f course there are several reasonable explanations for this discrepancy such as the probable differences in oncogen between the human breast tumors and the rat tumors, or the fact that in the human experiments the T u fractionation at 88°C yielded a 46% fraction of A + T rich D N A and in the rat experiments the TM fractionation was carried out at 91°C and yielded a 56.5% fraction of A + T rich DNA. However, still the most likely explanation for the varying neoplastic-associated disturbances observed between the rat and human, is probably correlated to differences in genomic distribution of (dA) regions. The data in Table 5 supports this argument. The (dA) regions in normal rat DNA are distributed equally into the G + C rich and main band DNAs but in the normal human genome they are not.
Acknowledgements--The
authors are indebted to Dr. Felix Mitelman for supply of transplanted DMBA tumors and to Dr. Albert Levan for his contributions to the genetic evaluation of some of the rat sarcomas.
REFERENCES 1. H. C. BIRNBOIM,R. E. J. MITCHEL and N. A. STRAUS, Analysis of long pyrimidine polynucleotides in Hela cell nuclear DNA: absence of polydeoxythymidylate. Proc. nat. Acad. Sci. (Wash.)70, 2189 (1973). 2. H. C. BIRNBOIM and N. A. STRAUS, DNA from eukaryotic cells contains unusually long pyrimidine sequences. Canad. 07. Biochem. 53, 640 (1975). 3. J. O. BISHOP, M. ROSBASH and D. EVANS, Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid. 07. mol. Biol. 85, 75 (1974). 4. S.T. CASE and R. F. BAKER, Detection of long eukaryotic specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos. 07. tool. Biol. 98, 69 (1975). 5. H. KUBINSKI, OPARA-KuBINSKA and W. SZYBALSKI, Patterns of interaction between polyribonucleotides and individual DNA strands derived from several vertebrates, bacteria and bacteriophages. 07. tool. Biol. 20, 313 (1966). 6. J. N. M. MOL and P. BORST, The binding of poly(rA) and poly(rU) to denatured DNA. II. Studies with natural DNAs. Nucleic Acids Res. 3, 1029 (1976). 7. R . W . PERO, T. BRYNGELSSON, C. BRYNGELSSON, A. DEUTSCH, A. NORDf~N and A. NORGREN, Hybridization of polyuridylic acid to human DNA immobilized onto nitrocellulose filters. Nucleic Acids Res. 2, 1163 (1975). 8. R. SEDEROFF, L. LOWENSTEINand H. C. BIRNBOIM,Polypyrimidine segments in Drosophila melanogaster DNA. II. Chromosome location and nucleotide sequence. Cell 5, 183 (1975). 9. A. SHENKIN and R. H. BORDON, Deoxyadenylate-rich sequences in mammalian DNA. FEBS Lett. 22, 157 (1972). 10. A. SHENKIN and R. N. BUROON,Deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA. 07. mol. Biol. 85, 19 (1974).
Polycyclic Hydrocarbon-Induced Rat Sarcomas 11. 12. 13.
14.
15. 16. 17.
18.
19.
20.
21.
22. 23. 24. 25. 26.
27.
28. 29.
30.
31.
N. A. STRAUS and H. C. BIRNBOIM,Long pyrimidine tracts of L-cell DNA: localization to repeated DNA. Proc. nat. Acad. Sci. (Wash.) 71, 2992 (1974). R. H. BURDON and A. SHENKIN,Uridylate-rich sequences in rapidly labelled RNA of mammalian cells. P~BS Lett. 24, 11 (1972). G . R . MOLLOY, W. L. THOMAS and J. E. DARNELL, Occurrence of uridylaterich oligonucleotide regions in heterogenous nuclear RNA of HeLa cells. Proc. nat. Acad. Sci. (Wash.) 69, 3684 (1972). H. NAKAZATO, M. EDMUNDS and D. W. KoPP, Differential metabolism of large and small poly(A) sequences in heterogeneous nuclear RNA of Hela cells. Proc. nat. Acad. Sci (Wash.) 71, 200 (1974). B. LEWlN, Units of transcription and translation: Sequence components of heterogeneous nuclear RNA and messenger RNA. Cell 4, 77 (1975). B. LEWIN, Units of transcription and translation, the relationship between heterogeneous nuclear RNA and messenger RNA. Cell 4, 11 (1975). R . W . PERO, T. BRYNGELSSON,A. NORGREN and A. DEUTSCH,Alterations in the deoxyadenylate regions of the DNA from four human breast tumors. Europ. J. Cancer 11, 861 (1975). R . W . PERO, T. BRYNGELSSON,A. DEUTSCH and ,~. NORD~N, Changes in the deoxyadenylate regions of DNA in 5 cases of chronic lymphocytic leukemia. Europ. J. Cancer 12, 357 (1976). R . W . PERO, T. BRVNOELSSON, F. MITELMAN and G. LEVAN, Changes in the deoxyadenylate regions of rat DNA in sarcomas induced by 7,12dimethylbenz (a )anthracene and Rous sarcoma virus. Hereditas 80, 153 (1975). T. T. CHEN and C. HEXBELBERGER, Quantitative studies on the malignant transformation of mouse prostate cells by carcinogenic hydrocarbons in vitro. Int. J. Cancer 4, 166 (1969). E. D. REES, Chromosomal aberrations in rat marrow cells following intravenous polycyclic hydrocarbons: possible basis for a carcinogen bioassay. In Proceedings in the Tobacco Health Workshop. Vol. 18, p. 42. University of Kentucky Tobacco Health Research Institute, Lexington (1969). G. LLwtx, U. AHLSTR6M and F. MITELMAN,The specificity of chromosome .\2 iuvohement in DMBA-induced rat sarcomas. Hereditas 77, 263 (1974). F. MITELMAN, Predetermined sequential chromosome changes in serial transplantation of Rous rat sarcomas. Acta path. microbiol, scand. 80, 313 (1972). J. MARMUR, A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. mol. Biol. 3, 208 (1961). J. WmcocKsoy, The use of sodium perchlorate in deproteinization during the preparation of nucleic acids. Biochem. J. 135, 559 (1973). S.Y. WAN% The determination of nucleic acid base composition by chemical reactivity. In Methods in Enzymology. (Edited by L. Grossman and K. Moldave) Vol. 12, part B, p. 178. Academic Press, New York (1968). G. LEVAN and A. LEVAN,Specific chromosome changes in malignancy: studies in rat sarcomas induced by two polycyclic hydrocarbons. Hereditas 79, 161 (1975). F. MITELMAN and G. LEVAN, The chromosomes of primary 7,12dimethylbenz(a)anthracene-induced rat sarcomas. Hereditas 71~ 325 (1971). M. DUNCAN,P. BROOKESand A. DIPPLE, Metabolism and binding to cellular macromolecules of a series of hydrocarbons by mouse embryo cells in culture. Int. J. Cancer 4, 813 (1969). T . J . SLAGA, S. G. BUTY, S. THOMPSON,W. M. BRACKENand A. VIAJE, A kinetic study on the in vitro covalent binding of polycyclic hydrocarbons to nucleic acids using epidermal homogenates as the activating system. Cancer Res. 37, 3126 (1977). T. GUTTMAN, H. VOTOVOV£ and L. PIVEC, Base composition heterogeneity of mammalian DNAs in CsCl-netropsin density gradient. Nucleic Acids Res. 3, 835 (1976).
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