Scand J Urol Nephrol9: 181-184, 1975

CREATINE PHOSPHOKINASE IN PROSTATIC TISSUE K. Sjovall, S. Rubin and J. Muntzing From the Research Laboraiories, AB Leo, Helsingborg, Sweden, and ihe Depariment of Urology. Universiiy of Lund, Lund, Sweden

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(Submitted for publication September 15, 1974)

Abstract. A histochemical study of creatine phosphokinase (CPK) in the rat ventral prostate revealed that the epithelial cells had a high cytoplasmic activity which was retained or even increased when the cells were atrophic as a consequence of castration. Both the fibromuscular stroma, which is prominent in the ventral prostate of castrated rats, and the secretion had a high level of activity. In human benign prostatic tissue where CPK was present in the epithelium, secretion and stroma. pronounced differences in activity could sometimes be observed between the epithelium of adjacent acini and also between cells in the same acinus. There was no obvious correlation between CPK activity and degree of epithelial hyperplasia. The activity in carcinomatous prostatic epithelial cells varied considerably between specimen from different patients but was uniform within a given specimen in contrast to the variation of epithelial CPK activity within non-carcinomatous tissue. Thus, a difference in the pattern of CPK activity was observed between malignant and nonmalignant epithelial cells.

I n the field of urology there has lately been interest in measuring CPK activity in serum following surgery of the bladder and prostate (Cattolica. 1971; Mulcahy, Greene & Connolly, 1971; Mahon, Muangman & Madsen, 1972) and in semen in connection with chronic prostatitis (Bostrom & Andersson, 1971). These latter authors found a high CPK activity in homogenates of the normal human prostate as well as in semen. CPK isoenzyme patterns in homogenates of benign and malignant prostatic tissue have also been reported (EIHilali, 1970). CPK activity has thus been found in homogenates of both benign and malignant human prostatic tissue. With a homogenization technique it is, however, impossible to determine the distribution of enzyme activity between the various tissue components. I n the present investigation a histochemical technique was used to study the distribution of CPK activity within the carcinomatous and noncarcinomatous human prostate. In addition the possible difference in CPK activity in normal and atrophic prostatic tissue from the rat was also investigated.

Phosphorylated creatine, creatine phosphate (CP), constitutes a readily available source of energy, especially in skeletal muscle (Morrison & Ennor, 1960). The high energy phosphate in CP is transferred to adenosine diphosphate (ADP) in a reaction catalysed by the enzyme ATP-creatine transphosphorylase (creatine phosphokinase, CPK, EC (Kuby & Noltmann, 1962). Thus, this enMATERIAL AND METHODS zyme has a key role in energy metabolism as it provides for a rapid resynthesis of adenosine tri- Ten male Wistar rats (SPF). 190-210 g, were obtained phosphate, the main metabolic energy source in the from Msllegaard's breeding laboratories. Ejby, Denmark. organism. A high level of CPK activity has been Half of the rats were castrated and half were sham operated. Twenty days after operation the animals were sacdemonstrated in muscular tissue both biochemically rificed and the ventral prostate was dissected out and (Kuby & Noltmann, 1962; Colombo, Richterich & weighed. The prostates were used for histochemical demRossi, 1962) and histochemically (Sjovall, 1%7; onstration of CPK by a nitro-blue tetrazolium (NBT) Khan, Holt, Knight & Kakulas, 1971) and determi- based method described by Sjovall (1967). The following modification in the fixation technique was found most nation of serum CPK activity is now a common suitable for the tissues investigated. The sections were laboratory test in patients with skeletal muscle dis- exposed for 30 minutes to an ice-cold 70% ethanol solution containing 10% fomalin. Control sections incubated eases or myocardial infarction. Scand J Urol Nephrol9

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K . Sjiivnll et ul.

Fig. 1 . The ventral prostate from an intact rat. A high CPK activity in the cytoplasm of the epithelial cells. x260.

Fig. 3 . Human benign prostatic hyperplasia. A high CPK activity in the cytoplasm of the epithelial cells. ~ 2 6 0 .

in the absence of the specific substrate-CP-showed little or no staining. Sections non-enzymatically stained with NBT showed a uniform staining (Sjovall, 1967). As Morton had reported in 1958 that a non-specific transphosphorylation from CP to glucose could take place in prostatic homogenates, sections were incubated without ADP but with CP in the otherwise complete reaction mixture. There was no staining in these sections. Human non-carcinomatous prostatic tissue was obtained from 6 patients at transurethral resection performed for urinary obstruction and from I I patients at transvesical prostatectomy performed for benign nodular hyperplasia. Specimens of carcinomatous prostatic tissue were obtained from twelve patients by perineal needle biopsy ad modum Veenema. Eight of the 12 patients had been treated for at least 2 months with estrogen or estramustine phosphate as described by Jonsson (1971) and Jonsson & Hogberg (1971). Four patients had not received any treatment when the biopsy were taken. The histochemical demonstration of CPK in human prostatic tissue was performed as described above.


Fi,c. 2 . The ventral prostate from a castrated rat. A high CPK activity in the cytoplasm of the epithelial cells. in the secretion and in the stroma around the acini. x2h0. Scctnd J Urn1 Nepltml9

In the normal rat prostate a positive histochemical reaction for CPK, a dark blue finely granular cytoplasmatic staining, was observed in the epithelial cells (Fig. I ). There was an accumulation of staining in the apical part of the cells. In the very thin fibromuscular stroma no positive CPK reaction could be observed. The secretion in the acini was dissolved during the histochemical procedure. Hence it was not possible to determine if CPK was present in the secretion from the ventral prostate of normal rats. The atrophic epithelial cells in the ventral prostate from the castrated rats showed a rather intense staining in the cytoplasm, indicating a high CPK activity (Fig. 2). A high activity was also found in the now more prominent fibromuscular stroma, especially around the acini where numerous immature smooth muscle cells are to be found (Tisell. 1970). The secretion in the small acini was often retained. This made it possible to establish that the secretion from the ventral prostate in castrated rats has a high CPK activity. CPK could usually be demonstrated in human non-carcinomatous prostatic tissue. However. in some specimens obtained by transurethral resection. no CPK activity could be seen. This was probably due to heat inactivation since these specimens showed signs of thermal injury. A high activity was found in both the stroma with its high content of smooth muscle cells and in the epithelial cells (Fig. 3). In the latter a concentration of staining was often seen in the apical areas. The CPK activity was not

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Creatine phosphokinase in prostatic tissue

Fig. 4 . H u m a n benign prostatic hyperplasia. Pronounced variation in epithelial CPK activity between different acini and also between cells in the same acinus. ~ 2 6 0 .

equally high in all epithelial cells. In some specimens the epithelial cells of one acinus had a much higher activity than those of an adjacent acinus. Pronounced activity variations could also be observed between epithelial cells of the same acinus (Fig. 4). There did not seem to be any correlation between the histochemically estimated activity of CPK and the degree of hyperplasia of the epithelium. In some sections small amounts of secretion had remained after preparation. This secretion had a high CPK activity. CPK could also be detected histochemically in carcinomatous tissue both in the stroma and in the malignant epithelial cells (Fig. 5). I n contrast to the results with non-malignant tissue the epithelial CPK activity was fairly uniform within sections from a given specimen. However, there was a considerable variation between specimens from the different patients. There was no obvious histochemical difference between the specimens from untreated and treated patients. Owing to the small number of patients it was not possible to correlate the intensity of CPK activity with the degree of tumour differentiation or with the effect of any previous anti-tumour treatment.


human prostate. The results from the present study and from the study of Bostrom & Andersson (1971) stand in contrast to the results obtained by Cattolica ( 1971) who found that prostatic homogenates contained no CPK activity. This discrepancy is probably due to differences in methodology. The apparent concentration of CPK activity in the apical part of the epithelial cells as well as the distinct CPK activity found in the acinar secretion is consistent with the CPK activity found in human semen (Lehmann & Griffiths, 1963: Bostrom & Andersson, 1971). An interesting finding is the observed cellular variation in CPK activity in some specimens of benign prostatic tissue. Such a variation in CPK activity between apparently similar cells was also observed among chondrocytes in growing long bones (Sjovall & Hansson, 1971) and it may reflect different metabolic states of the cells. However, this type of intercellular variation in CPK activity was not seen in the sections of malignant prostatic tissues which may indicate a less differentiated energy metabolism in the cancer cells. Most of the known enzymes in the rat ventral prostate show a decreased activity after castration (Price & Williams-Ashman, 1961). An increase induced by orchiectomy has only been reported for a few enzymes such as lactic dehydrogenase (Williams-Ashman, 1954), a-glycerophosphate dehydrogenase (Butler & Shade, 1958), aminopeptidase and P-glucuronidase (Andersson & Miintzmg, 1972). In the present study castration and concomitant prostatic atrophy did not 1t;sibly decrease

DISCUSSION The finding of a histochemically demonstrable high CPK activity in the epithelial cells of the human prostate corroborates the results of the biochemical study of Bostrom & Andersson ( I97 I ) who found a high CPK activity in homogenates of the normal

Fig. 5 . Human prostatic carcinoma. A high CPK activity in the cytoplasm of the malignant cells. x260. Scand J Urol Nephrol9

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K . Sjovall e t al.

the C P K activity in the epithelial cells. On the contrary, there was a n apparent increase in activity. CPK is present in homogenates of a number of malignant human tumours, such as carcinoma of the stomach, rectum and uterine cervix (Kleine, 1963, and also in prostatic carcinoma (EIHilali, 1970). The biochemical findings in the latter study were corroborated and extended in the present study where C P K could be localized to the cytoplasm of the malignant epithelial cells. The CPK activity varied markedly between the tumour samples from the different patients. In the small number of specimen examined in the present study there was n o obvious difference in CPK activity o r distribution between specimens from untreated patients and from patients treated for their disease. Studies of tissue specimens from further patients may elucidate a possible correlation between CPK activity and degree of tumour differentiation or effect of antitumour treatment.

R E FE R E NC ES Anderson, M. & Miintzing, J. 1972. The effect of a longacting estrogen on the activity and distribution of some hydrolases in the ventral prostate of intact, castrated, and androgen treated castrated adult rats. Invest Urol 9, 401. Bostrom, K. & Anderson, L. 1971. Creatine phosphokinase relative to acid phosphatase, lactade dehydrogenase, zinc and fructose in human semen with special reference to chronic prostatitis. Scand J Urol Nephrol5, 123. Butler. W. W. S. & Shade, A. L. 1958. The effects of castration and androgen replacement on the nucleic acid composition, metabolism and enzymatic capacities of the rat ventral prostate. Endocrinology 63, 271. Cattolica, E. V. 1971. Effect of transurethral surgery upon the serum enzyme creatine phosphokinase. J Urol106, 262. Colombo, J. P., Richterich, R. & Rossi, E. 1962. Serum- Kreatin-Phosphokinase: Bestimmung und diagnostische Bedeutung. Klin Wochenschr 40, 37. EIHilali, M. M. 1970. Enzymatic changes in prostatic carcinoma. Dissertation Abstr Intern 30. 3258.

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Jonsson, G. 1971. Treatment of prostatic carcinoma with pol yestradiol phosphate combined with ethinylestradiol. Scand J Urol Nephrol5, 97. Jonsson, G . & Hogberg, B. 1971. Treatment of advanced prostatic carcinoma with Estracytm. Scand J Urol Nephrol5, 103. Khan, M. A,, Holt, P. G., Knight, J. 0. & Kakulas, B. A. 1971. Incubation film technique for the histochemical localization of creatine kinase. Hisrochemie 26, 120. Kleine, T. 0. 1965. Die Aktivitat des Creatinkinase und anderer Enzyme des Kohlenhydrat-und Proteinstoffwechsels in benignen und malignen Tumoren des Menschen. Enzymuntersuchungen an menschlichen Tumoren I. Klin Wochenschr 43, 807. Kuby, S. A. & Noltmann. E. A. 1%2. ATP-creatine transphosphorylase. In The enzymes (ed. P. D. Boyer, H. Lardy & K. Myrback), 2nd ed., vol. 6, p. 515. Academic Press, New York and London. Lehmann, H. & Griffiths, P. D. 1%3. Creatinephosphokinase activity in semen. Lancet 11, 49. Mahon, F. B., Muangman, V. & Madsen, P. 0. 1972. Enzyme determinations after transurethral resection of prostate. J Urol107, 88. Morrison, J. F. & Ennor, A. H. 1960. N-phosphorylated guanidines. In The enzymes (ed. P. D. Boyer, H. Lardy & K. Myrback), 2nd ed., vol. 2, p. 89. Academic Press, New York and London. Morton, R. K. 1958. The phosphotransferase activity of phosphatases. 3. Comparison of enzymatic catalysis by acid phosphatase with non-enzymatic catalysis at acid pH values. Biochem J 70, 150. Mulcahy, J. J., Greene, L. F. & Conolly, D. C. 1971. Serum creatine phosphokinase levels following endoscopic urologic procedures. J Urol 105, 123. Price, D. & Williams-Ashman, H. C. 1961. The accessory reproductive glands of mammals. In Sex and internal secretion (ed. W. C . Young), vol. I , p. 366. Williams & Wilkins, Baltimore. Sjovall, K. 1%7. A tetrazolium technique for the histochemical localization of A T P creatine phosphotransferase. Histochemie 10. 336. Sjovall, K. & Hansson, L. J. 1971. The pattern of ATPcreatine phosphotransferase activity in growing long bones of newborn mice. Hisrochem J 3, 143. Tisell, L.-E. 1970. Effect of cortisone on the growth of the ventral prostate, the dorsolateral prostate, the coagulating glands and the seminal vesicles in castrated adrenalectomized and castrated nonadrenalectomized rats. Acta endocrinol (Kbh)64, 637. Williams-Ashman, H . G. 1954. Changes in the enzymatic constitution of the ventral prostate gland induced by androgenic hormones. Endocrinology 54, 12 I .

Creatine phosphokinase in prostatic tissue.

A histochemical study of creatine phosphokinase (CPK) in the rat ventral prostate revealed that the epithelial cells had a high cytoplasmic activity w...
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