Effect of Steroid Hormones on the in Vivo Incorporation of Glycine-2- 14C into Solid Ehrlich Tumor, Kidney, and Liver

Mitsuo Kodama and Toshiko Kodama Cancer Res 1970;30:228-235.

Updated Version

Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/30/1/228

E-mail alerts Reprints and Subscriptions Permissions

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org.

Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

[CANCER RESEARCH 30, 228-235,

January 1970]

Effect of Steroid Hormones on the in Vivo Incorporation of Glycine-2-14C into Solid Ehrlich Tumor, Kidney, and Liver
Mitsuo Kodama and Toshiko Kodama
Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, Nagoya, Japan

SUMMARY

The effect of steroid hormones for the nucleic acid and protein metabolism of solid Ehrlich tumor was studied by use of glycine-2-14C. The in vivo incorporation of glycine-14C into the nucleic acid and protein fractions of tumor tissue increased markedly 24 hr after the administra tion of 1 mg testosterone. Concomitantly, the increase of cellular constituents occurred successively in the order of RNA, protein, and DNA in the hormone-treated tumor as compared to the control tumor. The administration of 1 mg hydrocortisone acetate (HCA) resulted in a remarkable decrease in the incorporation of the tracer into tumor tissue 12 hr after the hormone injection. This inhibitive effect of HCA on tumor was more profound and persistent in male than in female mice, indicating that the hormone responsiveness of male tumor to HCA is much higher than that of female tumor. The decrease in the incorporation of glycine was also associated with the reduc tion of protein, RNA, and DNA contents of tumor tissue. Although there were some alterations in the tumor treated with estradiol, these changes were not in good accordance with the findings on the long-term experiment, in which the tumor growth was suppressed by the same hormone. It is indicated that the inhibitive effect of estradiol on tumor is a process requiring time and is to be distinguished from that of HCA. The differential actions of the three hormones were com pared in the liver, kidney, and solid tumor. The significance of these findings was discussed in the light of the molecular biology of steroid hormones.

and an inhibitive effect of both HCA and E on the growth of solid Ehrlich tumor. It was found that Ehrlich/4N tumor was more responsive than Ehrlich/2N tumor to the actions of T and HCA. The response of these tumors was expressed in terms of wet weight through the animal experiment. Because of the phenomenological nature of these data, the action of HCA was not differentiated from that of E in the suppression of tumor growth. This study was initiated to answer two questions. Is the action of a hormone on the tumor tissue specifically related to its chemical structure and/or to its physiological action? Is there any qualitative or quantitative difference in the action of a hormone from one tissue to the other? The effect of hormone administration was investigated by measuring (a) wet weight, (b) incorporation of glycine-2-14C into total nucleic acids and proteins, and (c) nucleic acid and protein contents of the solid tumor, kidney, and liver. The results obtained indicate that the actions of the 3 steroid hormones on the protein and nucleic acid metabolism of tumor tissue are different from each other, and that the response of tumor tissue to the administration of steroid hormones was contrasted to that of the liver and kidney to show the presence of tissue specificity in the action of each steroid hormone.
MATERIALS AND METHODS

The hypotetraploid Ehrlich ascites tumor, supplied by Dr. K. Kajiwara (Takeda Research Laboratory, Osaka, Japan), was used throughout this investigation. Solid tumors were produced by inoculating 5 X 10* tumor cells onto the backs of SMA mice. Gonadectomy was performed 1 month before tumor inoculation. All steroid hormones used in this study were suspended in 0.9% NaCl solution for intra muscular injection. Details of the animal experiment were described in the previous paper (28). The radioactive glycine-2-14C, with a specific activity of 9.9 mCi/mmole, was obtained from Dai-ichi Kagaku & Co., Ltd., Tokyo, Japan. In the experiment of amino acid incorporation in vivo, each mouse received i.p. l Ci of the labeled amino acid in a volume of 0.2 ml of 0.9% NaCl solution 12 hr before sacrifice. To accelerate the incorporation of labeled amino acid in vivo, the mice were starved for 12 hr after injection of radioactive glycine. The livers, kidneys, and tumors were each excised and stored in cold 10% trichloro acetic acid solution containing 0.4 M unlabeled glycine. The method of fractionation was originally based on the descrip-

INTRODUCTION

The investigation on the hormone responsiveness of Ehrlich ascites tumor was started by Kodama (26, 27) with the early observation that, when Ehrlich ascites tumor was inoculated subcutaneously, the mean weight of solid tumors thus developed was much greater in male than in female mice. The previous report (28) confirmed a stimulative effect of T1

'"The abbreviations used are: T, testosterone;

HCA, hydrocortisone

acetate; E, estradiol; TCA, trichloroacetic acid. Received April 11, 1968; accepted April 21, 1969.

228

CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Hormones and Ehrlich Tumor Metabolism tion of Schneider (38). The tissue was homogenized either in a Potter-Elvehjem homogenizer or in a Waring Blendor, and the added TCA solution was removed by centrifugation. The residue was washed once with cold 10% TCA to remove the free amino acid from the homogenate. It was then suspended in 6% TCA solution and incubated at 90for 15 min. After centrifugation the residue was washed twice with 6% TCA at room temperature. The first washing was combined with the 90extract to make a nucleic acid fraction. Aliquots of such fractions, after removal of TCA with ether, were plated directly in stainless steel planchis and dried under an infrared lamp. The remaining residue was incubated in ethanol-ether mixture (3:1) at 60 for 10 min. After centrifugation, the
RESULTS

Relation between the Weight Measurement and the Chemical Analysis. In order to determine whether the changes of tissue weight observed under various hormonal influences aie really associated with the changes in the content of cellular constituents, we made a comparative study with the liver, kidney, and solid tumor of SMA mice. Ehrlich/4N ascites tumor cells, 5 X IO6/ mouse, were

inoculated s.c. into 10 male and 10 female mice. The mice were sacrificed 12 days later. The livers, kidneys, and solid tumors were weighed and fractionated by the Schneider method for each mouse and for each tissue. As shown in Table 1, the mean weights of the 3 tissues are always greater precipitate was successively washed with ether, in male than in female mice, whereas there is not much chloroform:ether (4:1), and again ether. After the final difference between both sexes concerning the concentrations centrifugation, the residue was air dried and dissolved in of nucleic acid phosphorus and protein of these tissues. 0.05 N NaOH solution. Aliquots of this protein fraction Thus, the sex difference of these 3 tissues is equally were plated and dried similarly for counting. The radio significant with the wet weight as well as with the content activity was estimated in a windowless 2 vr gas flow counter of nucleic acid and protein (p < 0.001). In the tumor tissue with an automatic Aloka sealer; counts were corrected for each chemical measurement was converted into a common self-absorption. Activities of nucleic acid and protein samples logarithm which was in turn used for the ordinary statistical were expressed as cpm/mouse on one hand and as relative calculation, a mathematical rearrangement of data which was specific activity on the other hand. The relative specific found useful in the preceding paper for the statistical activity represents the ratio of the specific activity of analysis of tumor weight (28). A good proportionality exists hormone-affected tissue to that of a control tissue, the latter between the wet weight and the contents of nucleic acid and protein with the above 3 tissues. In the experiments being given as 1.000. The DNA content was estimated by the method of Burton hereafter the excised masses are weighed for each mouse and then pooled for each tissue and for each experimental group (7). Since the nucleic acid fraction in this study contained both RNA and DNA, RNA was estimated by the orcinol in the course of the Schneider fractionation. The statistical reaction (30) with a correction for the coexisting DNA. The analysis is to be limited to the data on wet weight of these measurement of phosphorus in the nucleic acid fraction was experiments. Effect of a Single Injection of T on the Fate of conducted by the method of Fiske and SubbaRow (11). Glycine-14 C in the Solid Tumor, Kidney, and Liver. The Before the colorimetrie reaction, samples were digested with 0.9 ml 60% perchloric acid in microkjeldahl flasks. The time course of hormonal effect after a single injection (1 phosphorus content of a nucleic acid sample was used for mg/mouse) of T was followed by measuring simultaneously the wet weight, the incorporation of glycine-14C, and the the calculation of specific activity of this fraction. Protein was determined by the procedure of Folin and Ciocalteu contents of nucleic acid and protein for each tissue. Cas trated SMA male mice were inoculated with 5 X IO6 tumor (12). In the colorimetrie reactions, calf thymus DNA, yeast RNA, and crystalline bovine serum albumin (all from Sigma cells on Day 0 and sacrificed on Day 14. The male hormone Chemical Co., St. Louis, Mo.) were used respectively as was given 12, 24, and 48 hr before sacrifice (a single standards for the determination of DNA, RNA, and protein. injection for each experimental group), and the radioactive Details of the statistical procedure were described in the glycine (1 /^Ci/mouse) was injected 12 hr before death. To previous paper (28). compare the hormonal response of the kidney and liver of

Table 1 Concentration of the protein and nucleic acid phosphorus in the liver, kidney, and solid tumor of protein," of nucleic acid phosphorus "x S.D. weight," xS.D. 7S.D. (mg)1930 HostMaleFemaleMaleFemaleMaleFemale"MeanTissueLiverLiverKidneyKidneyTumorTumorvalue wt.)853.3+ (Mg/g,wet (mg/g, wet wt.)165.1 3201349 129474 57304 351792+594289 128mice.Concentration of 10Wet 84.8867.0+ 48.2754.9 51.6881.1 101.4635.9 81.2575.0+ 55.2Concentration .8190.025 12.7156.9 7.4148.9+21.8111.7 9.2121.8 10.5

JANUARY

1970

229
Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Mitsuo Kodama and Toshiko Kodama tumor-bearing mice with that of tumor-free mice, the same time schedule of the hormone administration was applied for tumor-free castrated male mice as for the tumor-bearing ones. The mean tumor weight increased progressively with the lapse of time after the hormone administration, and the increase in tumor weight observed 48 hr after the hormone injection was statistically significant (0.001 < p < 0.01). Chart 1 summarizes the influence of T administration on the contents of nucleic acid and protein as well as on the The uptake of glycine-14 C by the kidney and liver of tumor-free mice was also increased by the hormone in jection, but this stimulative effect on the turnover of the amino acid was not associated with any substantial increase in the nucleic acid and protein contents of those tissues. The specific activity of 14C compounds of the nucleic acid and protein fractions was rather decreased by the same hormone in the livers of tumor-bearing mice (Chart 1, dotted-line graphs, left). In the kidneys of tumor-bearing mice the incorporation of glycine-14C was little affected by the male hormone (Chart 1, dotted-line graphs, middle^The discrep ancy between the findings of tumor-free mice and those of tumor-bearing mice might be explained by an interfering effect of the parasitic tumor for the metabolism of the host. Generally, T acts on the 3 tissues in such a way as to accelerate the turnover of nucleic acid and protein. In the case of the tumor tissue, however, the stimulative effect of the male hormone for the incorporation process is associated with the accumulation of the cellular constituents. Effect of a Single Injection of HCA on the Fate of Glycine-14 C in the Solid Tumor, Kidney, and Liver. The effect of a single injection of HCA on the 3 tissues was checked at regular times after the hormone injection. The experimental pro cedure is the same as the previous one except that HCA, 1 mg/mouse, was injected 12, 24, 48, and 72 hr before sacrifice (a single injection for each experimental group). A progressive decrease was observed after the hormone admin istration in the tumor weight, but not in the weights of kidney and liver. The suppressive effect of HCA was more prominent in male mice than in female mice. The difference in tumor weight was however statistically insignificant in both male and female mice. Charts 2 and 3 present the biochemical measurements of the 3 tissues in male and female mice. The administration of HCA to male mice resulted in a diminished incorporation'of glycine-14 C into tissue, which was observed in both the nucleic acid

II

n I s
I *
1234 Liver Protein 1234 Liver RNA 1234 Liver ON* 1234 Kidney Protein 1234 1234 Kidney Kidney RNA DNA 1234 Tumor Protein 1234 Tumor RNA 1234 Tumor ON

Chart 1. Effect of a single injection of T on the incorporation of glycine- C and on the nucleic acid and protein contents of the 3 tissues in castrated male mice. The male hormone, 1 mg/mouse, was administered 12 (Group 2), 24 (Group 3), and 48 hr (Group 4) before sacrifice respectively. The mice of Group 1 received no hormone injection. Each group in the tumor inoculation experiment consists of 18 to 21 mice, and the one in the tumor-free experiment consists of 10 to 12 mice. Group numbers are indicated below the abscissa; the ordinate for the histobar (left) represents percentage of tumor the corresponding value for Group 1; the ordinate for the line graph (right) denotes the relative specific activity of C compounds in the nucleic acid and protein fractions of each tissue. The data on the liver and kidney tissues of tumor-bearing mice are confined to the relative specific activity (dotted-line graphs).

incorporation

of glycine-14 C into the same fractions of liver,

kidney, and solid tumor. The former was expressed in terms of percentage of change with the histobars below, and the latter was arranged in the form of relative specific activity, as shown in the line graphs above. In accord with the change in tumor weight, the specific activity of 14C compounds in both nucleic acid and protein fractions of tumor increased by 19% and 44% respectively through the hormone treat ment. It is indicated that an accelerating effect of T for the uptake of glycine takes place first in the nucleic acid fraction of tumor tissue (12 hr after hormone treatment), and then ensues in the protein fraction (24 hr after hormone treatment). These data on the metabolism of tumor tissue was further fortified by the findings concerning the hor monal effect on the nucleic acid and protein contents, which increased by 48% (in protein and DNA contents) and 99% (in RNA content) following the hormone injection.

S
U

I o i
Chart 2. Effect of a single injection of HCA on the incorporation of glycine- C and on the nucleic acid and protein contents of the 3 tissues in male mice. The adrenocortical hormone, 1 mg/mouse, was administered 12 (Group 2), 24 (Group 3), 48 (Group 4), and 72 hr (Group 5) before sacrifice respectively. The mice of Group 1 received no hormone injection. Each group consists of 10 to 11 mice. The ordinales are as in Chart 1. The data on the liver and kidney tissues are all derived from the tumor-free groups.

230

CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Hormones and Ehrlich Tumor Metabolism contrast to the response of the male kidney, no initial decline of glycine incorporation was detected in the female kidney, and a remarkable rise of specific activity appeared at later stages of hormonal action. A minor increase was noted in the RNA content of female liver through the hormone treatment. The hormonal response of the liver and kidney of tumor-bearing mice, although not indicated in these charts, was essentially the same as that of tumor-free mice, but it seems that the presence of a solid tumor acts on the liver and kidney in such a way as to reduce the incorporation of glycine-14 C into those tissues.

l ooo

II

The decrease in the nucleic acid and protein contents of male tumor and the increase in the RNA content of male liver are the most prominent effects of HCA, although there Chart 3. Effect of a single injection of HCA on the incorporation of are a variety of insignificant and transient changes observed tissues. These findings are in good glycine- C and on the nucleic acid and protein contents of the 3 in other hormone-treated tissues in female mice. The experimental design for Chart 3 is the accordance with the fate of glycine-14 C as well as with the same as that for Chart 2. Each group consists of 9 to 12 mice. Group change in the wet weight of the corresponding tissues during numbers, with an addition of prime sign ('), are denoted as in the the hormone treatment. There is thus far no clue available to latter chart. Ordinales are also as in Charts 1 and 2. The data on the prove a possible relation between the function of female liver and kidney tissues are all derived from the tumor-free groups. liver and a relative resistance of female tumor to HCA. Effect of E Administration on the Fate of Glycine-14 C in Tumor-bearing Mice. The action of E was studied after the and protein fractions 12 hr after the hormone injection. The nucleic acid and protein contents of male tumor were also same experimental design as the previous one for HCA. The decreased by the hormone treatment. A fluctuation of the ovarian hormone, 1 mg/mouse, was administered i.m. to tumor-bearing male mice 12, 24, 48, and 72 hr before data observed 24 hr after the injection suggests that at least 2 kinds of proteins (and nucleic acids) with different rates of sacrifice (a single injection for each experimental group). There was no significant change in the wet weight of the 3 turnover, are involved in the expression of cortisone effect. tissues of hormone-treated mice. In the liver of tumor-free male mice, there was a transient The action of E in Chart 4. (but remarkable) enhancement of the incorporation of The specific activityonofthe 3 tissues is summarized fractions of 14C compounds in the 2 glycine-14 C into the nucleic acid fraction after the hormone tumor tissue was depressed slightly 12 hr after the hormone injection; the stimulation was less prominent in the protein fraction. The rise of the relative specific activity of liver injection, but this decrease in the specific activity was compensated by an increase in the cellular constituents at nucleic acid might be related to the increase in RNA content stage. The response of the liver and of liver. The response of the kidney in tumor-free male mice the corresponding was similar to that of tumor tissue, but the early fall of the kidney to the ovarian hormone is generally stimulative with
11345 Livr Protein 12345 Liver RNA 12345 Liver DNA 11345 1 2345 12345 Kidney Kidney Kidney Protein RNA DNA 12345 12345 Tumor Tumor Protein RNA 12345 Tjrrtor ONA 2 Q:

relative specific activity was followed by a rise of the same parameter at later stages, indicating that the action of HCA was counteracted by a homeostatic mechanism. Except for liver RNA, HCA administration did not affect the contents of cellular constituents. In female mice the incorporation of glycine into tumor tissue was also reduced by the hormone treatment, but the inhibition in female mice was less remarkable than in male mice, and a "rebound" phenomenon was observed at later stages in the protein fraction of tumor. The contents of the tumor constituents decreased 48 hr after the hormone injection, and increased remarkably in the subsequent 24 hr. It was indicated that the action of HCA on female tumor was of shorter duration when compared to the action on male tumor. As in the liver of a male mouse, a remarkable rise was observed in the specific activity of the nucleic acid fraction of female liver 12 hr after the hormone injection. Some increase was also noted in the specific activity of liver protein. The stimulative effect of HCA for the liver nucleic acid fraction was more remarkable in male than in female mice, whereas the increase in the specific activity of liver protein was more prominent in female than in male mice. In

A.

HI

12345 12345 Liver Liver Protetn RNA

12345 Liver DNA

HI
12345 Kidney Protein

12345 Kidney RNA

12345 Kidney DNA

12345 123*5 Tumor Tumor Protein RNA

III

12345 Tumor DNA

Chart 4. Effect on a single injection of E on the incorporation of glycine- C and on the nucleic acid and protein contents of the three tissues in tumor-bearing male mice. After the same time schedule as that for Chart 2, a single injection (1 mg/mouse) of the ovarian hormone was given to each group of mice. Each group consists of 13 to 15 mice. Group numbers and ordinales are denoted as in Chart 2. The data on the liver and kidney tissues are all derived from tumor-bearing mice.

JANUARY 1970

231
Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Mitsuo Kodama and Toshiko Kodama regard to the turnover of glycine-14C. The differences were however trivial or of shorter duration and were not asso ciated with any substantial increase of cellular constituents. For further investigation of the influence of E, the same hormone of varying doses (0.04 to 1.00 mg/day/mouse) was injected into tumor-bearing male mice for 4 consecutive days. Animals were sacrificed 24 hr after the last hormone injection. Other experimental designs are the same as the former 1-injection experiment. In spite of the repeated loading with E, the uptake of the tracer by the tumor tissue rather increased under the conditions used. Thus, the inhibitive effect of E on the tumor tissue, as revealed in terms of weight change, was not confirmed by the radioisotope experiment. It is supposed that the action of E on tumor tissue, a process requiring time, is elusive in the short-term experiment with the radioactive glycine. Effect of Various Steroid Hormones for the Efflux of 14C-Labeled Compounds from the 3 Tissues of Mice. The foregoing investigation was designed to check the effect of steroid hormones for the influx of glycine-14 C into various tissues (anabolic process of nucleic acid and protein metabo lism). The purpose of the last experiment is to follow the time course of the decline of radioactivity in the 14C-labeled tissues of mice with and without hormone treatment (catabolic process of nucleic acid and protein metabolism). The tumor-bearing mice were labeled in vivo by an injection of 1 /^Ci/mouse of glycine-2-I4C before starting hormone treatment. They were killed at intervals, and excised tissues were processed by the Schneider method for the determina tion of radioactivity of each fraction. As shown in Tables 2 and 3, the decline of radioactivity in tumor tissue of castrated male mice was considerably delayed by the admin istration of T. This reversing effect of T was observed in both the nucleic acid and protein fractions of tumor tissue, but it was less remarkable in the kidney protein. The decline of radioactivity in the liver protein was rather accelerated by T. The radioactivity of hormone-treated tumor was always higher than that of initial control (Group 1). The suppressive effect of T for the efflux of 14C compounds from tumor tissue might not be due to the retardation of catabolism, but to the acceleration of anabolism. The material required for the neosynthesis of tumor tissue could be mobilized from the liver and other tissues in view of the fact that the radioactivity of liver tissue was reduced appreciably by T treatment. The administration of HCA initially accelerates the efflux of 14C compounds from the tumor tissue (Group 2'), but this effect is no more detectable at a later stage (Group 3f). Likewise, some acceleration of the efflux was observed at the initial stage (Group 2") and some retardation of the efflux at the later stage of E treatment (Group 3"). In the liver and kidney, the initial accelerating effect for the efflux of 14C compounds, although less eminent than in the tumor tissue, was detected with HCA but not with E. These data indicate that the action of T is directed towards the stimulation of anabolism rather than the inhibition of catabolism, and that HCA and E exhibit a transitory acceleration of the efflux of 14C compounds from tumor tissue protein.
DISCUSSION

A number of reports have been published in the search for the mode of action of various steroid hormones on normal tissues. For solid Ehrlich tumor, possible participation of steroid hormones in tumor growth was first investigated by the biological measurements. Further dissection of the hormonal action was focused on the amino acidincorporating activity of target tissues on the basis of other investigations (3, 10, 13, 36). In the belief that the observa tion on the effect of a hormone should be made under

Table 2 Effect of steroid hormones for the efflux of 14, C-labeled compounds from the nucleic acid fractions of various tissues Castrated male mice were used in the experiment for testosterone, and intact male mice were used in the experiment for other hormones. They were inoculated with 1 /jCi/mouse of glycine-2- C i.p. on 9th inoculation day, and the hormones, 1 mg/day/mouse, were administered i.m. from Inoculation days 10 to 13, so that mice of Groups 2' and 2" received 1 injection, and mice of Groups 3' and 3" received 4 injections of hormone respectively. acidNo. of sacriLiver nucleic acidcpm/mouse81681881470710541246807810996768745961Relative nucleic nucleic acidRelative (inoculaRelative conof con con tiontentof14C1012121414101212121414143.10 day) cpm/mouse tent14C100100.099.786.6129.110064.765.080.061.659.977.1Tumor of cpm/mouse tent of 1 C1.62X mice1011111212101091011811fice IO32.63 X X1032.85 102.43 X IO32.67 X IO33.47 X IO31.47X IO32.16X IO31.46X IO31.99 IO34.91 X

Hormonal treatment1. None2. None2'. T3.

None3'.Tl.None2.

None2'. HCA2". E3. None3'. HCA3". EDate

IO32.57 X IO33.81 X IO32.81 X IO32.86 X IO33.25 X IO33.53 X IO32.18X X IO32.69 X IO32.18X IO33.34 X IO32.86 IO34.00 X X IO310084.991.878.386.110074.281.093.562.762.982.5Kidney X IO310091.2133.890.1122.810077.758.271.954

232

CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Hormones and Ehrlich Tumor Metabolism

Table 3. Effect of steroid hormones for the efflux of 14/ C-labeled compounds from various tissue proteins" Date of sacriLiver proteinfice (inoculaRelative conof tionC1012121414101212121414145.63 C day) cpm/mouse tent of mice1011111212101091011811Kidney treatmentl.None2. IO45.15 X IO44.20 X None2'. T3. IO43.58 X None3'. T1. None2. None2'. HCA2". E3. None3'. HCA3". ENo. " Values given as in Table 2. 103.44 X IO46.11 X proteinHormonal proteinTumor Relative content of.! C Relative content of '

cpm/mouse IO41.17X X IO41.20X IO40.70 IO40.78 X IO41.94X X

com/mouse IO46.41 X IO46.86 X IO44.05 X IO46.30 X IO41.63 X

IO45.80 X IO41.38 IO41.66X X IO45.14X X IO41.07 X IO41.04 IO41.35X X IO46.08 IO41.46 X IO43.28 X IO40.96 X IO40.89 IO42.95 X IO40.92 X IO40.96 X IO43.43 X IO40.96 X IO41.37X X X IO410091.574.763.861.210095.084.199.553.748.356.21.44 IO4100105.9118.970.2109.2100102.264.183.1 X IO410081.383.548.354.110071.255.375.549.247.449.25.77

conditions as natural as possible, the incorporation experi ment has been conducted at the level of the whole organism throughout this study. There was a good correlation among the measurements of wet weight, the incorporation of glycine-14C, and the

the 2 groups. In the case of tumor tissue, the contents of protein and nucleic acid had been replaced by their logarith mic values before the statistical calculation. Hence, a relative difference of 27.7% is to be a threshold value in the estimation of statistical significance with those parameters. contents of nucleic acid and protein in regard to the action The merit of the use of a radioactive tracer is that the time of T and HCA on the solid tumor (Charts 1 to 3). The course of hormonal action is followed in terms of the action of these hormones had been detected in the radio- relative specific activity, as shown in the action of T on the isotope experiment at an early stage before there appeared tumor tissue (Chart 1) or in the action of HCA on the any appreciable change in the wet weight or in the nucleic tumor and liver (Chart 2). Although there was no appre acid and protein contents. The same experiment indicated ciable change in wet weight as well as in the nucleic acid and that the response of liver and kidney to T was affected by protein contents of the kidney after HCA administration, the the presence of a solid tumor (Chart 1; Tables 2 and 3). The response of kidney to HCA as measured by the incorpora differential responsiveness of solid tumors to HCA in the tion of glycine-14C was quite similar to that of tumor. The male and female hosts was also demonstrated through the stimulative effect of T as observed in the tumor tissue could also be reproduced in the kidney through the long-term incorporation experiment. The long-term administration of E resulted in a decrease of administration (unpublished data). These findings indicate that tumor weight (28), but the suppressive effect of the the hormone-responsiveness of kidney is comparable to that hormone for tumor growth was not confirmed by the of the solid tumor. The liver makes quite a contrast to the other tissues in the incorporation experiment (Chart 4). The dissociation between the long-term experiment and the short-term experi expression of HCA action. As shown in this study, the 2 ment suggests that the expression of E action is not com tissues show quite opposite responses to T and HCA. The pleted merely by a primary rendez-vous of the hormone with mechanism of the tissue specificity as shown in the opposite actions of HCA on the tumor and on the liver, or of the the target cell but is mediated by other processes. A question arises as to whether a difference observed hormone specificity as evidenced in the antagonistic actions between the control and the hormone-treated groups is of T and HCA on the tumor, is to be elucidated by significant or not. Except for the first experiment, the reproducing the hormonal effect at the cellular level or at biochemical measurements were conducted each with a the subcellular level. The possibility that the specific affinity pooled sample, and no statistical analysis was applicable for of a target tissue for hormones is responsible for the those results. On the basis of the experiences with the first expression of hormonal action, was investigated by several experiment, it is possible to estimate the magnitude of workers (4-6, 25). There is, however, no evidence that the difference which is required to prove at the 95% confidence sensitivity of target cells to a steroid hormone is specifically that the 2 groups in question do not belong to one and the related either to the binding process or to the metabolic same universe. Suppose that the common standard deviation conversion of the hormone by the receptor cells. of the 2 groups takes a magnitude of 15% of the mean value In relation to the action of steroid hormones, the nucleic of the first group (for most of the data in the first acid and protein metabolism of the target cells was studied experiment, the S.D. did not exceed that value), and that with a number of cell-hormone associations. The accumu each group consists of 10 mice. Then, a relative difference of lated data indicate that the in vivo effect of steroid hor mones, whether it is stimulation or inhibition, involves the 14.1% in the mean value should be enough to differentiate

JANUARY

1970

233
Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Misuo Kodama and Toshiko Kodama process of nucleolar RNA synthesis (2, 14, 15, 29, 32). Amounts of actinomycin D which inhibited DNA-dependent RNA synthesis also prevented the hormonal responses (31, 33-35, 39). The proposed hypothesis that an early action of the hormone is to accelerate genetic expression was exam ined with a variety of steroid hormones in connection with the in vitro effect of hormones on the template activity of chromatin RNA (9, 37). The increase in the transcription capacity of chromatin DNA by the in vivo and in vitro action of a steroid hormone was also tentatively correlated to the physiological effect of the hormone (1, 8). However, some of the steroid hormone effects may be independent of the gene locus, as shown in the thymic involution with cortisone (16). It remains to be determined whether the hormone-gene thesis provides the molecular basis of steroid hormone actions. A neoplasm derived from a hormone-regulated tissue often retains the original hormone responsiveness, as shown in the inhibitive effect of cortisone on lymphatic leukemia or in the stimulative action of T on carcinoma of the prostate. In breast cancer, the situation is more complicated than in other malignancies. Various hormonal manipulations in cluding surgical treatments and administration of various steroid hormones were applied for controlling the progress of cancer. For elucidation of the mechanism of hormonal regulation of cancer, the experimental mammary tumor of rat were often used because of their relative hormone responsiveness. Hilf and his associates (1724) studied exten sively the biochemical characteristics of rat mammary tumors in relation to their hormone responsiveness. The tumors used were comparable to normal breast tissue in their biochemical and morphological responses to hormone treatment. The significance of hormone-responsive enzymes in the develop ment of these tumors remains to be elucidated. The present study on the solid Ehrlich tumor revealed that the 3 steroid hormones were involved each in different ways in the growth of the mouse tumor. It is hoped that a more complete information will be obtained concerning the hor monal aspect of cancer by use of the present tumor system.
ACKNOWLEDGMENTS We are grateful to Dr. Kazuo Ota for his continued interest and encouragement, and to Mrs. K. Yamauchi, Miss T. Takeda, and Miss T. Hanid.i for the technical assistance. 19. Breast Cancer. Brit. J. Cancer, 21: 714-726, 1967. 5. Braunsberg, H., and James, V. H. T. Observations on the Binding of Testosterone to Malignant Mammary Tumors and Other Tissues in Vitro. Brit. J. Cancer, 21: 703-713, 1967. 6. Burton, A. F. The Binding and Metabolism of Cortisol-4-C14 by Lymphatic Tissue and Tumors of Rats and Mice. Cancer Res., 24: 470-474, 1964. Burton, K. A Study of the Conditions and Mechanisms of the Diphenylamine Reaction for the Colorimetrie Estimation of Deoxyribonucleic Acid. Biochem. J., 62: 315-323, 1956. Dahmus, M. E., and Bonner, J. Increased Template Activity of Liver Chromatin, a Result of Hydrocortisone Administration. Proc. Nati. Acad. Sei. U. S., 54: 1370-1375, 1965. Dukes, P. P., Sekeris, C. E., and Schmid, W. On the Mechanism of Hormone Action. VI. Increase in Template Activity of Ribonucleic Acid from Isolated Nuclei Incubated in the Presence of Hormone. Biochim. Biophys. Acta, 123: 126-133,1966. Feigelson, P., and Feigelson, M. Studies on the Mechanism of Cortisone Action. In: G. Litwack and D. Kritchevsky (eds.), Action of Hormones on Molecular Processes, pp. 218-233. New York: John Wiley and Sons, Inc., 1964. Fiske, C. H., and SubbaRow, Y. The Colorimetrie Determination of Phosphorus. J. Biol. Chem., 66: 375-400, 1925. Folin, O., and Ciocalteu, V. On Tyrosine and Tryptophane Determinations in Proteins. J. Biol. Chem., 73: 627-650, 1927. Frieden, E. H. Sex Hormones and the Metabolism of Amino Acids and Proteins. In: G. Litwack and D. Kritchevsky (eds.), Actions of Hormones on Molecular Processes, pp. 509-559. New York: John Wiley and Sons, Inc., 1964. Gorski, J. Early Estrogen Effects on the Activity of Uterine Ribonucleic Acid Polymerase. J. Biol. Chem., 239: 889-892, 1964. Hancock, R. L., Zelis, R. F., Shaw, M., and Williams-Ashman, H. G. Incorporation of Ribonucleoside Triphosphates into Ribonu cleic Acid by Nuclei of the Prostate Gland. Biochim. Biophys. Acta, 55: 257-260, 1962. Hechter, O., and Halkerston, I. D. K. Effects of Steroid Hor mones on Gene Regulation and Cell Metabolism. Ann. Rev. Physiol., 27: 133-162, 1965. Hilf, R., Freeman, J. J., Michel, I., and Borman, A. Characteriza tion of a Transplantable Lactating Mammary Tumor: Endocrinological, Morphological, and Biochemical Aspects. Cancer Res., 24: 812-823, 1964. Huf, R., Goldenberg, H., and Bell, C. Effect of Actidione (Cycloheximide) on Estrogen-induced Biochemical Changes in R3230AC Mammary Tumors, Uteri, and Mammary Glands. Cancer Res., 27: 1485-1493, 1967. Hilf, R., Johnson, M. M., Breuer, C., Freeman, J. J., and Borman, A. Comparative Biochemistry of Three Transplantable Mammary Tumors as Influenced by Steroid Therapy. J. Nati. Cancer Inst., 31: 541-555, 1963. Hilf, R., Michel, I., and Bell, C. Dose Responses of R3230AC Mammary Tumor and Mammary Tissue to Estrogen: Enzymes, Nucleic Acids and Lipids. Cancer Res., 26: 865-870, 1966. Hilf, R., Michel, I., Bell, C., and Carrington, M. J. Influence of Endocrine Organ Ablation on the Growth and Biochemical Response of the R3230AC Mammary Tumor to Hormonal Treat ment. Cancer Res., 26: 1365-1370, 1966. Hilf, R., Michel, I., Bell, C., Freeman, J. J., and Borman, A. Biochemical and Morphologic Properties of a New Lactating Mammary Tumor Line in the Rat. Cancer Res., 25: 286-299 1965. Hilf, R., Michel, I., Silverstein, G., and Bell, C. Effect of Actinomycin D on Estrogen-induced Changes in Enzymes and Nucleic Acids of R3230AC Mammary Tumors, Uteri, and Mammary Glands. Cancer Res., 25: 1854-1859, 1965.

7.

8. 9.

10.

11. 12. 13.

14. 15.

16.

17.

18.

REFERENCES
1. Barker, K. L., and Warren, J. C. Effect of 17/3-Estradiol in Vitro on the Template Capacity of Uterine Chromatin. Endocrinology, 80: 536-539, 1967. 2. Barton, R. W., and Liao, S. A Similarity in the Effect of Estrogen and Androgen on the Synthesis of Ribonucleic Acid in the Cell Nuclei of Gonadohormone-sensitive Tissues. Endocrinol ogy, 81: 409-412, 1967. 3. Blecher, M., and White, A. Effect of Various Steroids and Metabolic Inhibitors on the Incorporation of Glycine-2-C14 into Total Proteins and Nucleic Acids of Normal and Malignant Lymphocytes in Vitro. J. Biol. Chem., 233: 1161-1168, 1958. 4. Braunsberg, H., Irvine, W. T., and James, V. H. T. A Comparison of Steroid Hormone Concentrations in Human Tissues Including

20.

21.

22.

23.

234

CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

Hormones and Ehrlich Tumor Metabolism

24. Hilf, R., Segaloff, A., and Lerner, L. J. Influence of Hormonal Treatment of Fischer Rats on the Biochemistry of a Transplantable Dimethylbenz(a)anthracene-induced Mammary Carcino ma and Its Sublines. Cancer Res., 28:, 1550-1558, 1968. 25. Hollander, N., and Yee, W. C. Relation between Cortisol Metabo lism and its Lympholytic Effect in P1798 Lymphosarcoma. Endocrinology, 79: 168-174, 1966. 26. Kodama, M. Effect of Hydrocortisone on Ehrlich Asches Tumor. Cancer Res., 22: 1212-1219, 1962. 27. Kodama, M. Influence of Gonads and Adrenals on Growth of Solid Ehrlich Tumor. Proc. Soc. Exptl. Biol. Med., 116: 1143-1147, 1964. 28. Kodama, M., and Kodama, T. Statistical Analysis of Hormonal Effects on the Steroid Responsiveness of Solid Ehrlich Tumor. Cancer Res., 30: 221-227,1970. 29. Liao, S., and Williams-Ashman, H. G. An Effect of Testosterone on Amino Acid Incorporation by Prostatic Ribonucleoprotein Particles. Proc. Nati. Acad. Sei. U. S., 48: 1956-1964, 1962. 30. Mejbaum, W. ber die Bestimmung kleiner Pentosemengen insbesondere in Derivaten der Adenylsure. Z. Physiol. Chem., 258: 117-120,1939. 31. Mishkin, E. P., and Shore, M. L. Inhibition by Actinomycin D of the Induction of Tryptophan Pyrrolase by Hydrocortisone. Biochim. Biophys. Acta, 138: 169-174, 1967. 32. Nakagawa, S., and White, A. Acute Decrease in RNA Polymerase Activity of Rat Thymus in Response to Cortisol Injection. Proc. Nati. Acad. Sei. U. S., 55: 900-904, 1966. 33. Nakagawa, S., and White, A. Response of Rat Thymic Nuclear RNA Polymerase to Cortisol Injection. Endocrinology, 81: 861-870, 1967. 34. Nicolette, J. A., and Mueller, G. C. Effect of Actinomycin D on the Estrogen Response in Uteri of Adrenalectomized Rats. Endocrinology, 79: 1162-1165, 1966. 35. O'Malley, B. W., McGuire, W. L., and Korenman, S. G. Estrogen Stimulation of Synthesis of Specific Proteins and RNA Poly merase Activity in the Immature Chick Oviduct. Biochim. Biophys. Acta, 145: 204-207, 1967. Pea,A., Dvorkin, B., and White, A. Effects of a Single Injection of Cortisol on Amino Acid-incorporating Activities of Rat Liver and Thymic Preparations In Vitro. .Biol. Chem., 241: 2144-2150, 1966. Schmid, W., Gallwitz, D., and Sekeris, C. E. On the Mechanism of Hormone Action. VIII. Further Studies on the Effect of Cortisol on Isolated Rat-Liver Nuclei. Biochim. Biophys. Acta, 134: 80-84, 1967. Schneider, W. C. Phosphorus Compounds in Animal Tissues. I. Extraction and Estimation of Deoxypentose Nucleic Acid and of Pentose Nucleic Acid. J. Biol. Chem., 161: 293-303, 1945. Ui, H., and Mueller, G. C. The Role of RNA Synthesis in Early Estrogen Action. Proc. Nati. Acad. Sei. U. S., 50: 256-260, 1963.

36.

37.

38.

39.

JANUARY 1970
Downloaded from cancerres.aacrjournals.org on February 23, 2012 Copyright 1970 American Association for Cancer Research

235