The Prostate 7:143-149 (1985)

Creatine Phosphokinase lsoenzymes in Human Prostatic Tissues: A Comparison Between Benign Hyperplasia and Adenocarcinoma Thomas Truong, Michel Carmel, Mostafa Elhilali, Denys Cloutier, and Jean-Guy Lehoux

Department of Biochemistry (7: T , J.G. L.), Gynecology and Obstetrics (D.C.), and Urology (M.C., M. E.), Faculty of Medicine, Sherbrooke University, Sherbrooke, PQ, Canada

The objective of this study was to determine the distribution of creatine phosphokinase (CPK) into its three isoenzymes, MM, MB, and BB, in human prostatic tissue, in patients with benign hyperplasia (BPH) and adenocarcinoma. Specimens were obtained from 23 patients with adenocarcinoma of the prostate and 25 patients with benign hyperplasia. We also had the opportunity to analyze the CPK content in two normal prostates, the first from a 16Yz-year-old boy and the second from a 9%-year-old child. Our results showed prostate tissue to contain almost exclusively the BB isoenzyme with traces of the MB and MM dimers in both cancer and BPH as well as the specimen of normal prostate from the 16%- year-old boy. As for the 9Yz-year-old child, we found the following distribution: 39% MM, 21 % MB, and 40% BB dimer. A comparison of the CPK-BB content in benign hyperplasia and adenocarcinoma revealed no significant difference between the two groups. Further- more, we tried to correlate prostatic tissue CPK-BB levels with another possible tumor marker of the prostate, prostatic acid phosphatase (PAP) measured in the cytosol. No correlation was found between these two markers. We also studied the relationship of CPK- BB and PAP content in prostatic tissue to nuclear and cytosolic androgen receptor content in human prostatic tissue. We found some correlation between CPK-BB and androgen cytosolic receptors as well as between PAP content and androgen cytosolic receptors in patients with benign hyperplasia. No such correlation was found in the group with adenocarcinoma.

In conclusion, this study does not show that the measurement of CPK-BB in the prostatic tissue could be used as an index of tissue malignancy.

Key words: human prostate, CPK isoenzyme, cancer, PAP, androgen receptor

INTRODUCTION

Creatine phosphokinase (CPK; EC 2.7.3.2) is a dimeric enzyme that catalyzes the reversible biochemical reaction, ATP + creatine = ADP + creatine PO4. It plays an important role in maintaining a readily available source of energy for cellular use as well as a key role in intracellular energy transport from mitochondria to sites of utilisation [ 13.

Received December 28, 1984; accepted February 8, 1985. M. Elhilali’s present address is Department of Urology, Royal Victoria Hospital, McCill University, Montreal, PQ, Canada. Address reprint requests to J.-G. Lehoux, Department of Biochemistry, Sherbrooke University, PQ, Canada J 1 H 5N4.

0 1985 Alan R. Liss, Inc.

144 Truongetal

There are three different isoenzymes of CPK: MM, MB, and BB. In skeletal muscle and myocardial tissue, the MM isoenzyme is found predominantly even though the latter also contains significant amounts of the MB. As for the BB isoen- zyme, the brain contains large amounts. Other tissues also rich in the BB dimer are the prostate, lung, urinary bladder, uterus, thyroid, pancreas, stomach, intestine, and kidney [2].

Since the early Seventies, the use of CPK as a biochemical marker of underlying pathological conditions has been studied [3-81. In some cases such as myocardial infarction and lesions of the central nervous system and skeletal muscle, its usefulness has been demonstrated [7]. In the field of cancer research, much interest in its use as a tumor marker has been shown, specifically for cancer of the prostate. In the past, most studies of CPK-BB have concentrated in detecting and quantifying the BB form in the serum of patients [3-61.

In this study, we have approached the problem at the tissue level, seeking to quantify CPK-BB in both prostatic cancer patients and those with BPH, to see if any significant difference existed. Furthermore, we tried to correlate the prostatic tissue CK-BB level with another possible tumor marker-namely, prostatic acid phosphatase (PAP)-and study their relationship to nuclear and cytosolic androgen receptor content.

MATERIALS AND METHODS Creatine Kinase lsoenzymes

The procedure we used was reported by Bailey [9]. Human tissue samples were obtained from patients by transurethral resection or open surgery and kept in liquid nitrogen until used. The two normal prostates were obtained from kidney donors at the time of kidney harvesting. From these samples, extracts were prepared by homogenizing the tissues in 10 volumes of a buffer containing 50 mM trisaminome- thane (TRIS), 0.25 M sucrose, 1 mM ethyleneglycol-bis-N,N’-tetraacetic acid (EGTA), and 1 mM DL-dithiothreitol (DTT) with a pH adjusted to 7.5 (at 23°C) with hydrochloric acid. The extracts were then centrifuged at 30,OOOg for 30 min at 4°C. After the first centrifugation, the supernatant was decanted and spun again for 30 min at the same speed. The final extract was thus obtained and kept on ice until separation on columns.

The columns consist of 5-cc Plastipak syringes filled with DEAE-Sephadex A- 50 anion exchange resin (Sigma Chemical Co, St Louis, MO) to a height of 1 cm. The resin was hydrated with a buffer containing 50 mM TRIS and 0.002% chlorhex- idine at pH 7.5. The eluting buffers used to separate the three CPK isoenzymes contain, respectively, 50 mM TRIS, 30 mM NaCl, pH of 7.5 for MM; 50 mM TRIS, 190 mM NaCl, pH of 7.5 for MB; and finally 50 mM TRIS, 400 mM NaC1, pH of 7.0 for BB.

Prior to separation, both column and buffers were warmed to room temperature. The column was then washed with 2 X 5-ml volumes of the MM eluting buffer. A 0.25-ml volume of tissue extract was applied to the top of the column. The elution of CPK-MM isoenzyme was obtained with the successive addition of MM buffer vol- umes of 1.2 and 3 ml, respectively. That of the CPK-MB isoenzyme was obtained with MB buffer volumes of 2 and 2 ml, and elution of CPK-BB isoenzyme with RB buffer volumes of 2 and 2 ml. All collected fractions were kept on ice until measure- ment of enzyme activity. Isoenzyme activity was measured at 30°C with the Rosalki

CPK in Human Prostatic Tissues 145

method [lo] using CPK-n-1 reagent (Worthington Diagnostics, Freehold, NJ) on a Varian Cary 219 spectrophotometer at 340 nm.

Nuclear and Cytosolic Androgen Receptor Assays

Androgen cytosolic and nuclear receptors were analyzed by the technique described by Hicks and Walsh et a1 [ 111. Radioactive samples were counted in a liquid scintillation counter (Model Beckman LS-900).

Prostate Acid Phosphatase (PAP) Assay

munoassay using commercial reagents (Beckon Dickinson, Orangesburg , NY) . The measurement of PAP was done in cytosol of prostatic tissue by radioim-

Protein and DNA Determination

The protein content was determined by the method of Lowry et a1 [12] with bovine serum albumin as standard. DNA was analyzed according to the method of Giles and Myers [13].

RESULTS

In prostatic tissue, BPH, and cancer, we found CPK-BB to be the dominant isoenzyme accounting for more than 90% of total activity. Only traces of CPK-MB and almost none of CPK-MM were found (Fig. 1). For the group of patients with BPH, the mean values for CPK content separated in its three isoenzymes, MM, MB, and BB, were, respectively, 0.00, 0.02, and 0.79 IU/mg of protein. The correspond- ing values of the group of patients with adenocarcinoma were 0.03, 0.02, and 0.95 IU/mg of protein. We found no significant difference in CPK-BB level between BPH

MM dimer

MB dimer

BB dirner

n

i C B NqN,

Fig. 1. Histogram showing distribution of CPK into its three isoenzymes-MM, MB, and BB- separated by column chromatography and measured enzymatically in prostatic tissue of patients with BPH (n = 25), adenocarcinoma of the prostate (n = 23). and two normal prostate specimens from 16%- and 9lh-year-old boys. C, adenocarcinoma; B, BPH; N1,16% years old; N2,9% years old.

146 Truongetal

and carcinoma (P = .27). The distribution of CPK into MM, MB, and BB dimers in the two normal prostate specimens of 16%- and 9Vz-year-old boys was, respectively, 0.02, 0.02, and 1.6 IU/mg of protein and 0.46, 0.24, and 0.47 IUlrng of protein.

Other parameters studied were androgen cytosolic and nuclear receptor content as well as prostate acid phosphatase (Tables I, 11). In the BPH group, we found mean values of 7.3 fmol/mg of protein for androgen cytosolic receptor content, 158.3 fmoll mg DNA for androgen nuclear receptor content, and 4.9 pglmg of protein for PAP. The corresponding values in the group of patients with adenocarcinoma were, respec- tively, 12.5 fmollrng of protein, 115.8 fmolhg DNA, and 7.2 pglrng of protein. Our study shows that there is no difference in cytosolic androgen receptor content between BPH and carcinoma (P = .069). Similarly, no difference in nuclear androgenic receptor content was noted (P = .18). There is also no difference in tissue PAP concentration between BPH and carcinoma (P = .26). These values in the normal prostate specimens were, respectively, 11.1 fmollmg of protein, 128.6 fmollrng DNA, and 3.4 pg/mg of protein for the 16E-year-old boy; 11.1 fmollrng of protein, 60.3 frnol/mg DNA, and 0.8 pglmg of protein for the 9%-year-old child.

Using a polynomial correlation regression test, we found there was some positive correlation between CPK-BB level and androgen cytosolic receptors in the BPH group (P < .05), and also between PAP content and androgen cytosolic receptors in this same group. No such correlation could be shown in the cancer group.

TABLE I. Comparison of the Concentration of Androgen Cytosolic and Nuclear Receptors in Patients With BPH and Adenocarcinoma in Human Prostate Tissue

Androgen cytosolic receptor Androgen nuclear receptors (fmol/mg protein) (fmol/rng DNA)

Cancer BPH Cancer BPH

Sample size 23 24 23 25 Mean 12.5' 7.3" I 15.8b 158.3' Std dev 11.0 1.9 99.3 116.9 Maxiniuni valuc 40.8 22.5 391.3 408.3 Minimum value 0.0 0.0 0.0 4.3 SEM 2.3 2.6 20.1 23.3

"t-test value, 1.86; associated P value, ,0690. 't-test value, 1.35; associated P value, 0.1841.

TABLE 11. Comparison of Prostate Acid Phosphatase Content in Human Prostate Tissue of Patients With BPH and Adenocarcinoma

Acid phosphatase (PAP) (pg/rng protein)

Cancer BPH

Sample size 21 20 Mean 7.2a 4.9a Std. dev. 1.5 4.1 Maximum value 24.3 20.6 Minimum value 0.3 0.8 SEM 1.6 1 .o 7-test value, 1.14; associated P value, .26

CPK in Human Prostatic Tissues 147

During our study, we noticed what seemed to be a difference in CPK-BB levels in patients with stage D carcinoma compared to those with less advanced disease (Fig. 2). Of our 23 cases in the group with adenocarcinoma, the distribution according to stage was 13 stage A2, one stage B1, three stage B2, one stage C, and five in stage D. Using a Krukal-Wallis test, the CPK-BB level in the stage D patients was found to be significantly lower (P = .02). All these stage D patients had poorly differentiated tumors and did not receive any hormonal treatment. No such differences between stage D patients and the other stages were found in the other studied parameters: androgen cytosolic and nuclear receptors.

DISCUSSION

The aim of our study was to find a tumor marker or a panel of tumor markers that would permit us to determine the degree of malignancy in prostatic tissue on one hand and on the other hand could eventually be used to monitor the response to various therapeutic approaches, whether it be hormonal or other.

The use of CPK-BB as a possible tumor marker for various types of cancer has been studied [3-81. Its possible use as a marker of adenocarcinoma of the prostate has been of particular interest. In the past, the studies of CPK-BB have been based mainly on its dosage in serum. Silverman et a1 [5] reported that 15 of 17 patients with untreated prostatic cancer had elevated CPK-BB serum values as compared to no significant elevation among 18 patients with prostatic cancer receiving and responding to chemotherapy. In a more recent study, Zweig et a1 [4] reported elevation of serum CPK in 29% of patients with adenocarcinoma of the prostate. In our study, we measured the CPK-BB concentration at tissue level in two groups of patients: one with BPH (n = 25) and the other with adenocarcinoma (n = 23). Our results indicate that there is no significant difference between these two groups. However, we did find a significantly lower content of CPK-BB in our patients with stage D carcinoma (n = 5) as compared to those with less-advanced disease (n = 18). In contrast, in studies in the serum, CPK-BB was found more often and in higher concentrations in

1.0

Y

m

1 D

Fig. 2. Histogram showing CPK-BB content measured enzymatically in prostatic tissue of patients with adenocarcinoma of prostate according to the stage of disease: A2 (n = 13); B, (n = 1); B2 (n = 3); C (n = 1); and D (n = 5) .

148 Truongetal

stage D adenocarcinoma of the prostate. A possible explanation for this finding may be that more CPK-BB is liberated into circulation in advanced disease state due to the anarchic metabolism of malignant cells with a concomitant liberation of CPK-BB from tissue. It would be interesting to see if this finding can be reproduced with a larger number of patients.

During our study, we had the opportunity to measure the CPK-BB content in two specimens of normal prostate. In the specimen from a 16%-year-old boy, we found a distribution of CPK into its three isoenzymes similar to that found in adults, whether it be BPH or adenocarcinoma, ie, predominance of the BB dimer with traces of MM and MB. In the specimen from the 9%-year-old boy, we found an isoenzyme distribution pattern quite different: MM 39%, MB 21%, and BB%. A possible explanation might be that with the hormonal changes associated with puberty, there is a shift in the CPK isoenzyme content of the prostate.

In studies in the serum, the dosage of PAP is much more useful and promising as a tumor marker [4]. In a study by Lmr et al [ 141, they found significantly lower quantities of PAP in tissues from patients with adenocarcinoma than in patients with BPH or normal prostates. In our study, we measured PAP in the cytosol of prostatic tissue and found no significant difference between BPH and cancer groups although we did also find lower mean values in the BPH.

We also measured the androgen receptor content in prostatic tissue. We found the androgenic receptor content, both nuclear as well as cytosolic, to be without significant difference in BPH and cancer. This finding confirms similar results in one of our previous studies [15,16] as well as those by other authors [17].

Finally in our study, we found some slight positive correlations between the CPK-BB content and the androgenic receptor content in the cytosol as well as between the PAP content and the androgenic cytosolic receptor content in the BPH group. These correlations were not found in the cancer group. This loss of correlation could be tentatively explained by the anarchic metabolism in malignant tissues not found in BPH .

In summary, we found that the dosage of CPK-BB, PAP, and androgenic receptors at the tissue level does not yield more information concerning the malignant state of disease in prostatic cancer than does the dosage of PAP and CPK-BB in the serum, particularly by the former.

ACKNOWLEDGMENTS

We thank very much Mrs. Lucie Du-Tremble who typed this manuscript and

This study was supported by a research grant from the National Cancer Institute Miss Francine Grondin for her technical assistance.

of Canada.

REFERENCES

1. Saks VA, Rosenshtraukh LV, Smirnov VN, Chazov EI: Role of creatine phosphokinase in cellular

2. Tsung SH: Creatine kinase isoenzyrne patterns in human tissue obtained at surgery. Clin Chem

3. Aleyassine H, MacIsaac SG: The diagnostic significance of serum creatine kinase BB isoenzyme in

function and metabolism. Can J Physiol Pharmacol 56:691-706, 1978.

22~173-175, 1976.

adenocarcinoma of prostate. Clin Biochem 13: 109-1 12. 1980.

CPK in Human Prostatic Tissues 149

4. Zweig MH, Van Steirteghem AC: Assessment by radioimmunoassay of serum creatine kinase BB as a tumor marker. Studies in patients with various cancers and a comparison of CK-BB concentra- tions to prostate acid phosphatase concentrations. JNCI 66:859-862, 1981.

5 . Silverman LM, Dermer GB, Zweig MH, Van Steirteghem AC, Tokes ZA: Creatine kinase BB: A new tumor-associated marker. Clin Chem 25: 1432-1435, 1979.

6. Silverman LM, Chapman JF, Jones ME, Dermer GB, Pullano T, Tokes ZA: Creatine kinase BB and other markers of prostatic carcinoma. The Prostate 2: 109-1 19, 1981.

7. Coolen RB, Pragay DA, Nosanchuk JS, Belding R: Elevation of brain-type creatine kinase in serum of patients with carcinoma. Cancer 44: 1414-1418, 1979.

8. Lederer WH, Gerstbrein HL: Creatine kinase isoenzyme BB activity in serum of a patient with gastric cancer. Clin Chem 22: 1748-1749, 1976.

9. Bailey WH: Ion-exchange chromatography of creatine kinase isoenzymes: A method with improved specificity and sensitivity. Biochem Med 24:3oO-3 13, 1980.

10. Rosalki SB: An improved procedure for serum creatine phosphokinase determination. J Lab Clin

11. Hicks LL, Walsh PC: A microassay for the measurement of androgen receptors in human prostatic tissue. Steroids 33:389-406, 1979.

12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the fohn phenol reagent. J Biol Chem 193:265-275, 1951.

13. Giles KW, Myers A: An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206:93, 1965.

14. Loor R, Wang MC, Valenzuela L, Chu TM: Expression of prostatic acid phosphatase in human prostate cancer. Cancer Lett 14:63-69, 1981.

15. Lehoux JG, Bdnard B, Elhilali M: Dihydrotestosterone receptors in human prostate. I. Nuclear concentration in normal, benign and malignant tissues. Arch Androl 5:237-248, 1980.

16. Elhilali M, Lehoux JG, Carmel M, Madarnas P, Mongeau C, Beauchesne C, Tdtreault L, Bdnard B: Nuclear androgen receptors of human prostatic tissue. A quantitative histological study. Arch Androl 10:21-27, 1983.

17. Shain SA, Boesel RW, Lamm DL, Rodwin HM: Cytoplasmic and nuclear androgen receptor content of normal and neoplastic human prostates and lymph node metastases of human prostatic adenocar- cinoma. J Clin Endocrinol Metab 50:704-711, 1980.

Med 69~696-705, 1967.