600 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 25 lO9

T H E M E T A B O L I S M I N V I T R O OF I4-1aC]CORTISONE

BY L Y M P H O S A R C O M A PI798

EDWARD CHANG*, ARNOLD MITTELMAN AND FRED ROSEN

Roswell Park Memorial Institute, BuOalo, N.Y. (U.S.A.)

(Received January 3Ist, 1964)

SUMMARY

The metabolism in vitro of [4-14C]cortisone by homogenates of cortisone-sensitive and resistant lines of lymphosarcoma PI798 was studied. The administration of cortisol for several days to mice bearing these tumors significantly altered the capacity of both tumor lines to metabolize cortisone. Following such treatment, the cortisone-sensitive tumor appears to be more efficient in converting cortisone to cortisol, while the resistant neoplasm is better able to convert cortisone to 2ofl-hydroxycortisone (REICHSTEIN'S substance "U") which is biologically inactive. The results suggest that the enzymes which mediate the formation of these metabolites may be responsive to glucocorticoid administration.

INTRODUCTION

The characteristics of a cortisone-responsive neoplasm, lymphosarcoma PI798, as well as the development of a cortisone-resistant line of this tumor have been described by MAcLAMPKIN AND POTTER 1. Experimental tissue of this type is useful for the study of various biochemical parameters that may be responsible for sensitivity and resistance to glucocorticoids. MAcLEoD, et al. ~ have recently reported that treatment with 9c~-fluoroprednisolone stimulates ribonuclease activity in the sensitive line of PI798 by as much as 7 ° %, whereas the level of ribonuclease activity in the resistant tumor was unaffected by the administration of this corticoid. Associated with the inhibition of growth of the sensitive tumor by cortisol, RosE~ AND NICHOL 3 observed a 9-fold rise in alanine-~-ketoglutarate-transaminase activity. Under the same conditions, the moderate reduction in growth of the cortisol-resistant PI798 tumor was not accompa- nied by any significant change in the activity of this transaminase.

The work of DOUGHERTY et al. 4 has contributed to an understanding of the metabolism of tile adrenal corticoids with respect to their influence on the growth and maturation of lymphocytes and lymphatic tissues. In this report, we wish to describe the metabolism of [4-1*C]cortisone when added in vitro to homogenates of cortisol- sensitive and resistant lines of lymphosarcoma PI798.

Present address: The Medical Foundation of Buffalo, 73 High street, Buffalo 3, N.Y. (U.S.A.).

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METABOLISM OF [4-14CIcoRTISONE BY LYMPHOSARCOMA 601

METHODS

Solid pieces (about 2 mm 3) of lymphosarcoma PI798 prepared under sterile conditions were transplanted by trocar into CDF (BALB/c >~ DBA/2) female mice about 6 weeks of age. The subcutaneous administration of cortisol (IOO mg/kg daily) for 2-4 days was started between the second and third week of tumor growth, when the tumors reached an average diameter of about 2 era. There were 5 animals in each group and they were killed 24 h after the last iniection of cortisol. Treatment with cortisol for 4 days pro- duced a substantial regression (about 9 ° %) of the cortisol-sensitive neoplasm, but less than a 5o % reduction in the size of the resistant tumor.

5 g of pooled tumor tissue from each group was homogenized in 20 ml of ice-cold o.25 M sucrose solution in a Virtis homogenizer operated at maximum speed for I rain. The entire homogenate was added to an incubation flask containing 5 /,moles ATP, 2.5/~moles TPN, 2.5/~moles DPN, 200/,moles Tris buffer (pH 7-4) and 6. 5- lO 4 counts/ rain of [4-14Clcortisone (specific activity 12.5 /,C/rag) in 0.05 ml of propylene glycol. The total volume of the incubation mixture was 25 ml. The flasks were incubated at 37 ° in an atmosphere of pure oxygen for 2 h in a shaking metabolic incubator.

After incubation, the mixture was extracted 4 times with 5 volumes of freshly distilled chloroform. The combined chloroform extracts were dried over NazSO 4 and then taken to dryness under vacuum. The residue was partitioned in a mixture con- taining equal volumes of IOO ml each of heptane and 7 ° % aqueous methanol. Approx. 8o-85 % of the added radioactivity was recovered in the 7o % methanol fraction. The radioactive compounds in this fraction were separated by paper chromatography.

Whatman No. I paper, previously washed with methanol, was equilibrated over- night in a closed tank with a mixture of equal volumes of toluene and 7 ° 5~, aqueous methanol. The samples and appropriate radioactive standards were chromatographed for 6 h at 3 o°. A 2-cm wide strip of the chromatogram was scanned for radioactivity with a Scanogram I I I (Atomic Accessories Inc.) at a speed of 6 in/h. When the radio- active areas with peak counts were located, they were cut out and eluted with ethanol. The ethanol was removed under vacuum and the residues further identified by counter- current distribution.

The methods used for counter-current distribution and assay of radioactivity were previously describedS.The solvent systems were similar to those used by CARSTENSEN 6. Following the counter-current separation, the contents of the tubes containing peak radioactivity for each fraction were pooled and the residues were acetylated in a mixture of pyridine and acetic anhydride. The acetate derivatives were chromato- graphed on Whatman No. I paper, prepared as described above, but in a chloroform- formamide system.

RESULTS

Paper chromatography (TM-7o) of the extracts from the cortisol-sensitive (Fig. I) and resistant (Fig. 2) lines of lymphosarcoma PI798 incubated with E4-14Clcortisone re- vealed several peaks of activity. The compounds present in each of these areas were identified, while the polar material which remained at the origin has not been char- acterized.

The radioactive compound at peak I was provisionally identified as 4-pregnene- I7~-2o/3, 2I-triol-3,II-dione, REICHSTEIN'S substance "U" . The eluate of t is area was

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602 E. CHANG, A. MITTLEMAN, F. ROSEN

mixed with I mg of standard substance "U" as carrier and the mixture submitted to counter-current distribution in 75 % ethyl acetate-25 % n-hexane/Ioo % water system (System A) for 25 transfers. The partition coefficient (K = o.71 ) calculated from the

FRONT k ~ S 4 - 'm " ~ ' t ' - S 4 °]I. ""kS4-1"l ORIGIN

FRONT N---S 2-'m" ,t, S2-.I].",'k- $ 2 - I--d ORIGIN

FRONT I,- SQ_-]II-,I I'SO-I]-,+,'-- S O - I --,,I ORIGIN

Fig. i. Metabolites of cortisone obtained by incubation of the cortisone-sensitive t umor with F4-14C]cortisone. So, tumors obtained from untreated control animals; $2, after t r ea tment with

cortisol for 2 days and $4, after four injections of cortisol.

radioactive curve was identical to that of the added standard which was estimated spectrophotometrically at 240 m/~. Paper chromatography of the acetate derivatives of the radioactive compound and the carrier indicated essentially the same Ry value of o.6o-o.62.

The following results indicate that cortisol is the compound located at peak II . Counter-current distribution of the eluted radiochemical with added standard cortisol was carried out in 5 ° % ethyl acetate-5o % n-hexane/Ioo % water system (System B) for 25 transfers. The partition coefficient (K = I.O) of the radioactive material was similar to that of the added cortisol which was measured spectrophotometrically at 28o m/,. Chromatography of the acetate derivatives revealed similar Rv values of o.5I-O.53 for the radioactive compound and cortisol diacetate.

Peak I I I : this peak corresponds to unchanged cortisone. The partition coefficient (K = 1.4o) of the radioactive compound in system B and the RF value (o.84) of its acetate derivative were identical to that obtained with a pure sample of cortisone.

Biochim. Biophys. Acta, 90 (z964) 600-605

METABOLISM OF [4-14C]coRTISONE BY LYMPHOSARCOMA 603

The data shown in Figs. I and 2 and Table I indicate that treatment of the tumor- bearing animals with cortisol altered the tumor's capacity for metabolizing cortisone in vitro. After treatment with cortisol for 2 days, the conversion of cortisone to

FRONT h , . ~ R 4 - 'tTr H I,,- R 41I-, 'k- R 4 1 --,'1 ORIGIN

FRONT " k - R 2 -TIT - - - , I H ~ 2 - ] I - d , - - R Z - I ~ d ORIGIN

- - IOOO C O U N T S / M I N U T E

FRONT I.- R 0 - '~=--.I I~O-~.d*-RO - I --d ORIGIN

Fig. 2. Metabolites of cortisone obtained by incubation of the cortisone-resistant t u m o r with [4-14C]cortisone. Ro, t umors obtained f rom unt rea ted control animals; R2, after t r ea tmen t wi th

cortisol for 2 days and 1R4, after four injections of cortisol.

T A B L E I

METABOLISM OF E4-14C]CORTISONE BY LYMPHOSARCOMA PI798

Treatment Reichstein' s "" U" (I) Cortisol (II) Cortisone (III)

(%)* (Counts/rain)** (%) (Counts/min) {%) (Counts/rain)

Sens i t i ve t um or

None 37.0 189 320 2. 7 13 81o 60.0 307 52o IOO mg cortisol/kg daily (2 days) 16.6 lO 4 ooo 5.3 33 725 78.2 489 85 ° lOO mg cortisol/kg daily (4 days) 7.1 29 650 19.2 78 45 ° 73.5 3 °2 525

Res i s tan t t u m o r

None 34.1 163 16o 3.2 15 320 64.5 298 050 ioo mg cortisol/kg daily (2 days) 19.7 lO 3 ioo 3.8 19 8o0 76.5 399 25o ioo mg cortisol/kg daily (4 days) 12.2 76 42o 11. 7 61 730 74.0 390 825

* Relative percentage of the total eluted radioact ivi ty in Peaks I, I I and I I I . ** Radioact iv i ty eluted f rom the area under the curve of the chromatogram.

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604 E. CHANG, A. MITTLEMAN, F. ROSEN

REICHSTEIN'S substance "U" was reduced to about 5o % of the control value for each tumor. This effect was more pronounced when cortisol was given for 4 days and under these conditions the sensitive line appears to have less capacity of forming substance "U", than the resistant tumor. An increase in the conversion of cortisone to cortisol in vitro occurred with both tumor lines, obtained from animals pretreated with cortisol. About a 2-fold increase in the amount of cortisol formed was noted in the homogenate of the sensitive tumor following treatment with 2 doses of cortisol, and after 4 injections of cortisol the amount of cortisone converted to cortisol was 6 times greater than that observed for the untreated tumor homogenate. In this regard, the resistant tumor did not respond after two doses of cortisol but about a 4-fold rise in the amount of cortisol formed was observed in the system in vitro following 4 daily injections of cortisol.

DISCUSSION

The results of this study demonstrate the capability of homogenates of lymphosarcoma PI798 to metabolize [4-14Clcortisone, The spectrum of metabolites obtained from both tumor lines were qualitatively similar. However, the cortisone-sensitive tumor seems to be more efficient in converting cortisone to 2ofl-hydroxycortisone (REICHSTEIN'S substance "U"). This quantitative difference between the tumors was not observed unless the tumors were obtained from animals that were treated with cortisol.

The alterations in the metabolism of [4-14C] cortisone which were observed in both tumor lines after treatment with cortisol, suggest that an increase in the activity of Ilfl-hydroxy-dehydrogenase and a decrease in 2ofi-hydroxy-dehydrogenase activity occurred. Direct measurement of these enzymes in both lines of lymphosarcoma PI798 , however, is required to demonstrate that they are responsive to cortisol.

I t was previously suggested that the differences between the two lines of PI798 might be explained on the basis of impaired uptake of glucocorticoids by the cortisone- resistant line 7. This possibility now seems unlikely, since in this study treatment with cortisol altered the capacity of both tumors to metabolize cortisone.

Lymphocytokaryorrhectic activity of cortisol, but not of cortisone, has been described by DOUGHERTY et al. 4. Thus, the regression of a cortisone-sensitive tumor following treatment with cortisone probably involves the conversion of this corti- costeroid to its Ilfl-hydroxyanalogue, or cortisol. However, the fact that the sensitive line of PI798 is responsive to other corticoids such as cortisol and 9~-fluoroprednisolone, suggests that I ifi-hydroxy-dehydrogenase need not play an important role in mediating the response of this tumor to glucocorticoids. The formation of REICHSTEIN'S sub- stance "U" by the resistant line of PI798 may, in part, explain the lack of effect of cortisone on the growth of this neoplasm, since compound "U" does not possess lymphocyte suppressing activity.

In all of the chromatographic separations and in both tumor lines, approximately the same amount of adrenosterone was collected in the solvent run-off during the preliminary chromatography. A small amount of II-fl-hydroxyandrostenedione was found in the untreated, cortisone-resistant sample.

There is increasing support for the concept that the hormonal control of the structure and function of certain tissues is mediated by altering the activity of specific enzymesS, 9. In this regard, the ability of a hormone to affect the level of an enzyme(s)

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METABOLISM OF [4-14C]CoRTISONE BY LYMPHOSARCOMA 6 0 5

concerned with its own activation or inactivation may be an important aspect of the specificity of hormone action in target tissues.

The lack of any significant difference in the metabolism of F4-14C1 cortisone between the two tumor lines prior to t rea tment of the tumor-bearing mice with cortisol is not readily explained. The resistant form of PI798 was developed by exposure of the sensitive line to cortisone during numerous transplant generations. Such t reatment might be expected to result in constitutive biochemical or physical changes in the cell that would be related to resistance to corticosteroids. The present study did not reveal such differences and further studies are required to determine the significance of the increased conversion of cortisone to its inactive analogue, 2ofl-hydroxycortisone, that occurs in vitro in the resistant tumor following t reatment with cortisol.

ACKNOWLEDGEMENTS

The authors wish to express their thanks to Dr. D. BERLINER for a generous supply of REICHSTEIN 'S substance "U" and to Mrs. E. VOLTIG for conscientious technical assistance.

This work was supported in part by a grant from United Health Fund of Western New York (G-63-RP-9-I).

REFERENCES

1 j . MAcLAMPKIN AND M. POTTER, ,/. Natl. Canc. Inst., 20 (1958) lO91. 2 R. M. MACLEoD, C. E. KING AND V. P. HOLLANDER, Cancer Res., 23 (1963) lO45. 3 F. ROSEN AND C. A. NICHOL, Proc. Am. Assoc. Cancer Res., 3 (1961) 263. 4 T. F. DOUGHERTY, M. L. BERLINER AND D. L. BERLINER, in L. M. TOCAUTINS, Progress in

Hematology, Vol. 3, Grune a nd S t r a t t on , New York, 1962, p. 155. 5 E. CHANG, A. MITTELMAN AND T. DAO, J. Biol. Chem., 238 (1963) 913 . 6 H. CARSTENSEN, in D. GLICK, Methods of Biochemical Analysis, Vol. 9, In tersc ience , New York ,

1962, p. 127. 7 F. ROSEN, Cancer Res., 23 (1963) 1447. 8 D. E. GREEN, Adv. Enzymol., I (1941) 177. 9 H. A. KREBS, H. KORNBERG AND I~. A. BURTON, Ergeb. Physiol., Biol. Chem. Exptl. Pharmakol.,

49 (1957) 212.

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