- Email: [email protected]
Template capacity of chromatin from normal and neoplastic tissues
Template capacity of chromatin from normal and neoplastic tissues JAMES C. WARREN, M.D., PH.D.* ¥,.ENNETH L. BARKER, PH.D,** Kansas City, Kansas The capacities of purified chromatin preparations from neoplastic and adjacent normal tissues to serve as a template for RNA synthesis were compared in vitro. In the malignant neoplasms studied, the template capacity of tumor chromatin per microgram of DNA was less than that of chromatin from normal tissue. Total chromatin obtained from tumor tissue (on the basis of DNA content) was 1.4 to 4 times that from normal tissue. It is concluded that chromatin from these neoplasms is not extensively "de-repressed" and on the basis of DNA content is actually repressed as template for RNA synthesis.
I T r s N o w well established that DNA serves as a template in the synthesis of RNA by DNA-dependent RNA polymerase. Bonner and Huang1 purified chromatin and described a method to evaluate its template capacity (template activity) by assay with labeled nucleotides and DNA-dependent RNA polymerase from E. coli. Marushige and Bonner2 demonstrated that stripping of histones from rat liver chromatin results in DNA which has a fivefold greater template capacity and suggested that histones inhibit transcription. Previous studies in this laboratory have indicated that the uterine growth which occurs after administration of estradiol-17.B to the ovariectomized rat 3 or during the
estrogen surge in the intact cycling hamster 4 is associated with an increase in the template capacity of uterine chromatin. Because disturbance of growth control regulatmy mechanisms seems to be characteristic of neoplasms, it seemed pertinent to evaluate the capacity of chromatin prepared from tumor and nontumor tissues to serve as a template for DNA-dependent RNA synthesis. This communication reports the results of that evaluation. Materials and methods
Tumor and adjacent areas of nontumor tissue were obtained at surgical extirpation and immediately frozen in liquid nitrogen. Only nonnecrotic tumor tissue was used. Absence of necrosis was assured grossly and by microscopic sections. Preparation of purified chromatin. Chromatin was prepared by the procedure of Marushige and Bonner2 utilizing modifications previously described by Barker and Warren~ to allow preparation from 0.5 Gm. of tissue. The tissue was homogenized in 30 ml. of saline-EDTA (0.05M NaCI and 0.16M Na2 EDTA, pH 8.0) and 0.15 ml. of 2-octanol in a VirTis Model 23 homogenizer for 1 minute at 110 volts and 4 minutes at 70 volts. The homogenate was strained
From the Departml!'nts of Obstetrics-Gynecology and Biochem 'stry, University of Kansas School of Medicine. This research was supported by United States Public Health Service Research Grant AM-05546. *Career Development Awardee, National Institute of Child Health and Human Development. **Postdoctoral Trainee, National l>nstitute of Child Health and Human Development. Present address: Department of Obstetrics and Gynecology, University of Nebraska College of Medicine, Omaha, Nebraska.
198
Volume 99 Number 2
through two layers of Miracloth and centrifuged at 1,500 x g for 15 minutes. The sedi= ment was washed with 12 ml. of salineEDT A, and 12 mi. of Tris buffer (0.05M, pH 8.0) followed each time by centrifugation at 1,500 x g for 15 minutes. The sediment was suspended twice in 5 ml. of Tris buffer (0.05M, pH 8.0) by aspiration through a small-bore pipette and centrifuged at 10,000 x g for 15 minutes. The pellet was taken up in 5 ml. of Tris buffer ( 0.05M, pH 8.0), stirred for 30 minutes, and layered over 25 ml. of 17M sucrose in Tris buffer (O.OlM, pH 8.0). The upper two-thirds was gently mixed and the preparation centrifuged at 30,000 x g for 2 hours. The gel-like pellet was washed twice in 10 mi. of Tris buffer (O.OlM, pH 8.0) and centrifuged after each wash at 30,000 x g for 30 minutes. The final pellet was suspended in Tris buffer (0.01M, pH 8.0). DNA content was determined and the chromatin was assayed immediately, in duplicate, for template capacity. All preparative steps were carried out at 0-4° C. Assay of template capacity. The reaction mixture for RNA synthesis was in accord with the conditions specified by Bonner and Huang1 and contained in a final volume of 0.25 mi.: 10 p.moles Tris bufl"er (pH 8.0), 1.0 ,umole MgClz, 0.25 ,umole MnC1 2, 3.0 ,umoles ,8-nlercaptoethanol, 0.1 p.mole each of cytidine 5'-triphosphate, uridine 5'-triphosphate and guanosine 5'-triphosphate, 0.1 ,umole (1 ,000 c.p.m. per millimicromole) of 8- 14Cadenosine 5'-triphosphate, chromatin equivalent to 12 ,ug of DNA and purified DNAdependent RNA polymerase. The enzyme was purified from early log phase cells of E. coli strain B (General Biochemicals) by the method of Chamberlin and Berg5 to the stage of their Fraction 3. Following incubation at 37° C. for 10 minutes, the reaction was stopped by the addition of cold 6 per cent trichloroacetic acid (TCA). Acid-insoluble material was collected on membrane filters (Schleicher and Schuell, B-6) and washed seven times with 3 ml. portions of cold 3 per cent TCA. The filters were glued to aluminum planchets, dried and counted in a
Templote capacity of chromatin
199
Nuclear-Chicago windowless gas flow counter. 3 Sufficient counts vverc collected to give a counting error not exceeding 2 per cent. DNA determinations. An aliquot of purified chromatin was hydrolyzed in 0.5M perchloric acid at 70° C. for 15 minutes. The hydrolysate was cooled and centrifuged at 2,000 x g for 15 minutes. The optical density of the supernatant was measured at 260 mp., corrected for light scattering as measured at 320 m,u, and DNA concentrations were obtained by comparison with a standard DNA solution. The concentration of DNA in the preparation was verified the following day using the diphenylamine 6 reaction and was always within 5 per cent of the amount determined by the ultraviolet absorption method. Results Before conducting the actual experiment it was necessary to evaluate possible effects of freezing. This was done by securing a fresh liver from a 180 gram female rat of the Holtzman strain, dicing it and separating the fragments into equal portions. One portion was frozen in liquid nitrogen while preparation of chl'omatin was started immediately from the second. Three hours later, the frozen portion was thawed and chromatin prepared in an identical manner. Both preparations Vv"ere assayed simuttane . . ously in duplicate in the system described above. With chromatin from the unfrozen sample (equivalent to 12 p.g DNA), 1,580 p.J.tmoles of adenosine 5'-monophosphate (AMP) were incorporated into RNA while with chromatin from the frozen sample (equivalent to 12 /kg DNA) 1,584 tJ.p..'Tioles were incorporated. It was concluded that freezing had no effect on the template capacity. The results of the template capacity assay (expressed as microinicromoles of AMP incorporated into RNA per 10 minut-es) of normal and neoplastic tissues are shown in Table I. In only one case does the template capacity (per microgram of DNA) of the neoplastic tissue exceed that of the normal and this is not necessarily significant as dif-
200
Warren and Barker
September 15, 1967 Am. J. Obst. & Gynec.
Table I. Template capacity of chromatin from normal and neoplastic tissue* AMP
Patient No.
2 3
Tissue
Colon mucosa Adenocarcinoma of colon Normal ovary (left) Arrhenoblastoma (right) Omentum Adenocarcinoma, t metastatic to omentum
incorporated (p,p.moles)
1,507 1,217 260 287 1,090 434
4
Cerv·ix
830
5
Squamous carcinoma of cervix Myometrium Mixed mesodenna! tumor
643 637 299
Normal tissue (%)
81 109 40
Chromatin extracted (DNA in P.!!l c.c. tissue)
290 960 296 1,200 330 850
Total template capacity/ c.c. tissue as % of normal tissue
370 448 103
1A(I
"'V
78
600 1,280
333
47
1,640
61
*Chromatin was purified and assayed for template capacity as described in Methods with 12 p.g of DNA in the form of purified chromatin used in all instances. The added RNA polymerase was capable of incorporating 8,465 p.p.moles AMP per 0.25 mi. when 50 p.g of purified salmon sperm DNA was used as primer and incorporation without any primer (284 p.p.moles AMP per 0.25 mi.) has been subtracted from the values reported. tThe adenocarcinoma in Patient No. 3 had primary origin in the colon.
ferences in duplicate assays were 5 to 8 per cent. In all other cases, template capacity of the tumor tissue chromatin is significantly less ( 40 to 8i per cent of normal). It is of interest that the total quantity of DNA in the form of chromatin obtained from tumor tissues was 1.4 to 4 times more than that obtained from a block of normal tissue of approximately the same size. While these values must be taken as only approximate because of possible variations in recovery, they do suggest somewhat more chromatin per unit volume of tissue, a not too surprising finding. Based upon these values the total template capacity per unit volume of tissue ([,up.rnoles AMP incorporated/,ug DNA] x [,ug DNA/c.c. tissue]) can be calculated and normal and neoplastic tissues compared as in Table I. In all but one instance, total template capacity per cubic centimeter of the neoplastic tissue is equal to or exceeds normal tissue. Comment
Bardos and associates 7 have shown that the capacity (per microgram of DNA) of DNA from neoplastic tissues to serve as template for DNA synthesis is increased approximately twofold over that of normal tissues. Further, the yield of DNA per gram weight of tissue was greater from neoplastic
tissues. The DNA studied by Bardos and associates/ however, was not in the form of purified chromatin but rather had been submitted to extensive deproteinization as described by Marmur8 and exceeded even purified calf thymus DNA in its capacity as a template to direct DNA synthesis. Studies on in vivo hepatic RNA synthesis have indicated that while regeneration 9 of tissue is associated with RNA synthesis, so is starvation/0 indicating that stimulation of growth is not a single common denominator. Similar in vivo studies have indicated that excluding pancreas, RNA synthesis is usually higher, 11 • 12 but sometimes lower 12 in tumor than in normal tissues. Possibly the over-all capacity to synthesize extensive amounts of RNA is not as important to cellular division as to differentation and assumption of metabolic duties. In vivo RNA synthesis depends upon availability of nucleotides, activity of RNA polymerase and available template. The results obtained in this investigation pertain only to the template factor as nucleotide concentrations are essentially saturating and variations in endogenous RNA polymerase activity are negligible in comparison to amounts added. Therefore, a direct comparison of these results with those obtained in vivo is not warranted.
Volume Y9
\lumber 2
The data do indicate that chromatin from 'de-repressed." The arrhenoblastoma was a 'unctional androgen producing tumor which acked morphologic criteria of malignancy; :he other 4 specimens were clearly malig1ant. The malignant tumors appear to be :per microgram of DNA) clearly repressed ts template for RNA synthesis. Conclusions as to the total template ca>acity per unit volume of tissue are not 1uite as clear cut. In three of the four in;tances where tumor chromatin can be :ompared with that from tissue at the site >f tumor origin total template capacity per mit volume of tumor tissue is two to three imes that of normal tissue. In the fourth, a nixed mesodermal tumor, it is less. While
REFERENCES l. Bonner, J., and Hua..Ylg, R. C.: Biochem. Biophys. Res. Commun. 22: 211, 1966. 2. Marushige, K., and Bonner, J.: J. Molec. Bioi. 15: 160, 1966. 3. Barker, K. L., and Warren, J. C.: Proc. Nat. Acad. Sc. 56: 1298, 1966. 4. Warren, J. C., and Barker, K. L.: Bioehim. biophys. acta 138: 421, 1967. 5. Chamberlin, M., and Berg, P.: Proc. Nat. Acad. Sc. 48: 81, 1962. 6. Burton, K.: Biochem. J. 62: 315, 1956. 7. Bardos, T. J., Ambrus, J. L., Chmielewicz, Z. F., Penny, A. G., and Ambrus, C. M.: Cancer Res. 25: 1238, 1965. 8. Marmur, J. A.: J, Molec. Bioi. 3: 208, 1961. 9. Ultman, J. E., Hirschberg, E., and Gellhorn, A.: Cancer Res. 13: 14, 1953.
Template capacity of chromatin
201
it is tempting to speculate that this follows from the fact that the rnixed mesodermal tumor displays lower quantities of DNA per unit volume because of the presence of stromal nonnuclear elements, variation in recovery is also a possibility. Because of this possible variation in recovery, and because the methodology used in this study does not distinguish between the types of RNA (transfer, messenger, ribosomal, or nuclear) being synthesir.ed, possible implications of the finding that template capacity per microgram of DNA is decreased in the chromatin of malignant neoplasms cannot be fruitfully applied to the concepts of "biochemical uniformity" 1 r~ and "reduced enzyme template lifetime." 14
10. Cantarov1_, A.. , Pac~ki&, K. E., and \\qniaffis, T. L.: Bioehem. Biophys. Res. Commun. 1: 75, 1959. 11. .Brue$, A. M., Tracy, M. M., and Cohn, W. E.: J. Bioi. Chem. 155t619, 1944. 12. Heidelberger, C., Leibman, K. C., Barbers, E., and Bhargara, P. M.: Cancer Res. 17: 399, 1957. 13. Greennein, J. P.: Cancer Res. 16: 611, 1956. 14. Pitot, H. C.: Perspect. Bioi. & Med. 8: 50~ 1964.
Rainbow at 39th Street Kansas City,~ Kansas 66103
I T r s N o w well established that DNA serves as a template in the synthesis of RNA by DNA-dependent RNA polymerase. Bonner and Huang1 purified chromatin and described a method to evaluate its template capacity (template activity) by assay with labeled nucleotides and DNA-dependent RNA polymerase from E. coli. Marushige and Bonner2 demonstrated that stripping of histones from rat liver chromatin results in DNA which has a fivefold greater template capacity and suggested that histones inhibit transcription. Previous studies in this laboratory have indicated that the uterine growth which occurs after administration of estradiol-17.B to the ovariectomized rat 3 or during the
estrogen surge in the intact cycling hamster 4 is associated with an increase in the template capacity of uterine chromatin. Because disturbance of growth control regulatmy mechanisms seems to be characteristic of neoplasms, it seemed pertinent to evaluate the capacity of chromatin prepared from tumor and nontumor tissues to serve as a template for DNA-dependent RNA synthesis. This communication reports the results of that evaluation. Materials and methods
Tumor and adjacent areas of nontumor tissue were obtained at surgical extirpation and immediately frozen in liquid nitrogen. Only nonnecrotic tumor tissue was used. Absence of necrosis was assured grossly and by microscopic sections. Preparation of purified chromatin. Chromatin was prepared by the procedure of Marushige and Bonner2 utilizing modifications previously described by Barker and Warren~ to allow preparation from 0.5 Gm. of tissue. The tissue was homogenized in 30 ml. of saline-EDTA (0.05M NaCI and 0.16M Na2 EDTA, pH 8.0) and 0.15 ml. of 2-octanol in a VirTis Model 23 homogenizer for 1 minute at 110 volts and 4 minutes at 70 volts. The homogenate was strained
From the Departml!'nts of Obstetrics-Gynecology and Biochem 'stry, University of Kansas School of Medicine. This research was supported by United States Public Health Service Research Grant AM-05546. *Career Development Awardee, National Institute of Child Health and Human Development. **Postdoctoral Trainee, National l>nstitute of Child Health and Human Development. Present address: Department of Obstetrics and Gynecology, University of Nebraska College of Medicine, Omaha, Nebraska.
198
Volume 99 Number 2
through two layers of Miracloth and centrifuged at 1,500 x g for 15 minutes. The sedi= ment was washed with 12 ml. of salineEDT A, and 12 mi. of Tris buffer (0.05M, pH 8.0) followed each time by centrifugation at 1,500 x g for 15 minutes. The sediment was suspended twice in 5 ml. of Tris buffer (0.05M, pH 8.0) by aspiration through a small-bore pipette and centrifuged at 10,000 x g for 15 minutes. The pellet was taken up in 5 ml. of Tris buffer ( 0.05M, pH 8.0), stirred for 30 minutes, and layered over 25 ml. of 17M sucrose in Tris buffer (O.OlM, pH 8.0). The upper two-thirds was gently mixed and the preparation centrifuged at 30,000 x g for 2 hours. The gel-like pellet was washed twice in 10 mi. of Tris buffer (O.OlM, pH 8.0) and centrifuged after each wash at 30,000 x g for 30 minutes. The final pellet was suspended in Tris buffer (0.01M, pH 8.0). DNA content was determined and the chromatin was assayed immediately, in duplicate, for template capacity. All preparative steps were carried out at 0-4° C. Assay of template capacity. The reaction mixture for RNA synthesis was in accord with the conditions specified by Bonner and Huang1 and contained in a final volume of 0.25 mi.: 10 p.moles Tris bufl"er (pH 8.0), 1.0 ,umole MgClz, 0.25 ,umole MnC1 2, 3.0 ,umoles ,8-nlercaptoethanol, 0.1 p.mole each of cytidine 5'-triphosphate, uridine 5'-triphosphate and guanosine 5'-triphosphate, 0.1 ,umole (1 ,000 c.p.m. per millimicromole) of 8- 14Cadenosine 5'-triphosphate, chromatin equivalent to 12 ,ug of DNA and purified DNAdependent RNA polymerase. The enzyme was purified from early log phase cells of E. coli strain B (General Biochemicals) by the method of Chamberlin and Berg5 to the stage of their Fraction 3. Following incubation at 37° C. for 10 minutes, the reaction was stopped by the addition of cold 6 per cent trichloroacetic acid (TCA). Acid-insoluble material was collected on membrane filters (Schleicher and Schuell, B-6) and washed seven times with 3 ml. portions of cold 3 per cent TCA. The filters were glued to aluminum planchets, dried and counted in a
Templote capacity of chromatin
199
Nuclear-Chicago windowless gas flow counter. 3 Sufficient counts vverc collected to give a counting error not exceeding 2 per cent. DNA determinations. An aliquot of purified chromatin was hydrolyzed in 0.5M perchloric acid at 70° C. for 15 minutes. The hydrolysate was cooled and centrifuged at 2,000 x g for 15 minutes. The optical density of the supernatant was measured at 260 mp., corrected for light scattering as measured at 320 m,u, and DNA concentrations were obtained by comparison with a standard DNA solution. The concentration of DNA in the preparation was verified the following day using the diphenylamine 6 reaction and was always within 5 per cent of the amount determined by the ultraviolet absorption method. Results Before conducting the actual experiment it was necessary to evaluate possible effects of freezing. This was done by securing a fresh liver from a 180 gram female rat of the Holtzman strain, dicing it and separating the fragments into equal portions. One portion was frozen in liquid nitrogen while preparation of chl'omatin was started immediately from the second. Three hours later, the frozen portion was thawed and chromatin prepared in an identical manner. Both preparations Vv"ere assayed simuttane . . ously in duplicate in the system described above. With chromatin from the unfrozen sample (equivalent to 12 p.g DNA), 1,580 p.J.tmoles of adenosine 5'-monophosphate (AMP) were incorporated into RNA while with chromatin from the frozen sample (equivalent to 12 /kg DNA) 1,584 tJ.p..'Tioles were incorporated. It was concluded that freezing had no effect on the template capacity. The results of the template capacity assay (expressed as microinicromoles of AMP incorporated into RNA per 10 minut-es) of normal and neoplastic tissues are shown in Table I. In only one case does the template capacity (per microgram of DNA) of the neoplastic tissue exceed that of the normal and this is not necessarily significant as dif-
200
Warren and Barker
September 15, 1967 Am. J. Obst. & Gynec.
Table I. Template capacity of chromatin from normal and neoplastic tissue* AMP
Patient No.
2 3
Tissue
Colon mucosa Adenocarcinoma of colon Normal ovary (left) Arrhenoblastoma (right) Omentum Adenocarcinoma, t metastatic to omentum
incorporated (p,p.moles)
1,507 1,217 260 287 1,090 434
4
Cerv·ix
830
5
Squamous carcinoma of cervix Myometrium Mixed mesodenna! tumor
643 637 299
Normal tissue (%)
81 109 40
Chromatin extracted (DNA in P.!!l c.c. tissue)
290 960 296 1,200 330 850
Total template capacity/ c.c. tissue as % of normal tissue
370 448 103
1A(I
"'V
78
600 1,280
333
47
1,640
61
*Chromatin was purified and assayed for template capacity as described in Methods with 12 p.g of DNA in the form of purified chromatin used in all instances. The added RNA polymerase was capable of incorporating 8,465 p.p.moles AMP per 0.25 mi. when 50 p.g of purified salmon sperm DNA was used as primer and incorporation without any primer (284 p.p.moles AMP per 0.25 mi.) has been subtracted from the values reported. tThe adenocarcinoma in Patient No. 3 had primary origin in the colon.
ferences in duplicate assays were 5 to 8 per cent. In all other cases, template capacity of the tumor tissue chromatin is significantly less ( 40 to 8i per cent of normal). It is of interest that the total quantity of DNA in the form of chromatin obtained from tumor tissues was 1.4 to 4 times more than that obtained from a block of normal tissue of approximately the same size. While these values must be taken as only approximate because of possible variations in recovery, they do suggest somewhat more chromatin per unit volume of tissue, a not too surprising finding. Based upon these values the total template capacity per unit volume of tissue ([,up.rnoles AMP incorporated/,ug DNA] x [,ug DNA/c.c. tissue]) can be calculated and normal and neoplastic tissues compared as in Table I. In all but one instance, total template capacity per cubic centimeter of the neoplastic tissue is equal to or exceeds normal tissue. Comment
Bardos and associates 7 have shown that the capacity (per microgram of DNA) of DNA from neoplastic tissues to serve as template for DNA synthesis is increased approximately twofold over that of normal tissues. Further, the yield of DNA per gram weight of tissue was greater from neoplastic
tissues. The DNA studied by Bardos and associates/ however, was not in the form of purified chromatin but rather had been submitted to extensive deproteinization as described by Marmur8 and exceeded even purified calf thymus DNA in its capacity as a template to direct DNA synthesis. Studies on in vivo hepatic RNA synthesis have indicated that while regeneration 9 of tissue is associated with RNA synthesis, so is starvation/0 indicating that stimulation of growth is not a single common denominator. Similar in vivo studies have indicated that excluding pancreas, RNA synthesis is usually higher, 11 • 12 but sometimes lower 12 in tumor than in normal tissues. Possibly the over-all capacity to synthesize extensive amounts of RNA is not as important to cellular division as to differentation and assumption of metabolic duties. In vivo RNA synthesis depends upon availability of nucleotides, activity of RNA polymerase and available template. The results obtained in this investigation pertain only to the template factor as nucleotide concentrations are essentially saturating and variations in endogenous RNA polymerase activity are negligible in comparison to amounts added. Therefore, a direct comparison of these results with those obtained in vivo is not warranted.
Volume Y9
\lumber 2
The data do indicate that chromatin from 'de-repressed." The arrhenoblastoma was a 'unctional androgen producing tumor which acked morphologic criteria of malignancy; :he other 4 specimens were clearly malig1ant. The malignant tumors appear to be :per microgram of DNA) clearly repressed ts template for RNA synthesis. Conclusions as to the total template ca>acity per unit volume of tissue are not 1uite as clear cut. In three of the four in;tances where tumor chromatin can be :ompared with that from tissue at the site >f tumor origin total template capacity per mit volume of tumor tissue is two to three imes that of normal tissue. In the fourth, a nixed mesodermal tumor, it is less. While
REFERENCES l. Bonner, J., and Hua..Ylg, R. C.: Biochem. Biophys. Res. Commun. 22: 211, 1966. 2. Marushige, K., and Bonner, J.: J. Molec. Bioi. 15: 160, 1966. 3. Barker, K. L., and Warren, J. C.: Proc. Nat. Acad. Sc. 56: 1298, 1966. 4. Warren, J. C., and Barker, K. L.: Bioehim. biophys. acta 138: 421, 1967. 5. Chamberlin, M., and Berg, P.: Proc. Nat. Acad. Sc. 48: 81, 1962. 6. Burton, K.: Biochem. J. 62: 315, 1956. 7. Bardos, T. J., Ambrus, J. L., Chmielewicz, Z. F., Penny, A. G., and Ambrus, C. M.: Cancer Res. 25: 1238, 1965. 8. Marmur, J. A.: J, Molec. Bioi. 3: 208, 1961. 9. Ultman, J. E., Hirschberg, E., and Gellhorn, A.: Cancer Res. 13: 14, 1953.
Template capacity of chromatin
201
it is tempting to speculate that this follows from the fact that the rnixed mesodermal tumor displays lower quantities of DNA per unit volume because of the presence of stromal nonnuclear elements, variation in recovery is also a possibility. Because of this possible variation in recovery, and because the methodology used in this study does not distinguish between the types of RNA (transfer, messenger, ribosomal, or nuclear) being synthesir.ed, possible implications of the finding that template capacity per microgram of DNA is decreased in the chromatin of malignant neoplasms cannot be fruitfully applied to the concepts of "biochemical uniformity" 1 r~ and "reduced enzyme template lifetime." 14
10. Cantarov1_, A.. , Pac~ki&, K. E., and \\qniaffis, T. L.: Bioehem. Biophys. Res. Commun. 1: 75, 1959. 11. .Brue$, A. M., Tracy, M. M., and Cohn, W. E.: J. Bioi. Chem. 155t619, 1944. 12. Heidelberger, C., Leibman, K. C., Barbers, E., and Bhargara, P. M.: Cancer Res. 17: 399, 1957. 13. Greennein, J. P.: Cancer Res. 16: 611, 1956. 14. Pitot, H. C.: Perspect. Bioi. & Med. 8: 50~ 1964.
Rainbow at 39th Street Kansas City,~ Kansas 66103