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Regional distribution of ornithine decarboxylase activity and polyamine levels in experimental cat brain tumors
Neurochemistry International 39 (2001) 135– 140 www.elsevier.com/locate/neuint
Regional distribution of ornithine decarboxylase activity and polyamine levels in experimental cat brain tumors Gabriele Ro¨hn a, Thomas Els b, Karen Hell c, Ralf-Ingo Ernestus a,* b
a Department of Neurosurgery, Uni6ersity of Cologne, Cologne, Germany Department of Experimental Neurology, Max-Planck-Institute for Neurological Research, Cologne, Germany c Department of Pathology, Uni6ersity of Cologne, Cologne, Germany
Received 2 June 2000; received in revised form 3 January 2001; accepted 8 January 2001
Abstract Biosynthesis of the polyamines putrescine, spermidine, and spermine, and activation of the first key enzyme ornithine decarboxylase (ODC) are closely associated with cellular proliferation. In the present study, the distribution of ODC activity and polyamine levels was investigated for the first time regionally in experimental brain tumors of the cat. Brain tumors were produced by stereotactic xenotransplantation of rat glioma cells. Twenty days after implantation, the brains were frozen in situ, cut into slices, and cryostat sections and tissue samples were taken to determine ODC activity and polyamine levels biochemically. The quantified data were color-coded to present the regional distribution of ODC activity and polyamine levels in the respective section. ODC activity significantly increased in some areas within the tumor, whereas peritumoral tissue showed no difference to the non-tumoral, contralateral hemisphere. This increase turned out in parallel to a high number of mitoses in the same tumor parts (r =0.861). Putrescine levels increased both, in the whole tumor and in the peritumoral edema. Regional differences in putrescine content did not correlate with solid and proliferative parts of the tumor. Spermidine and spermine levels were only slightly increased in some parts of the tumor. Thus, these experiments show the close correlation of a high mitotic rate and activation of ODC within experimental gliomas and underline the relevance of ODC as a biochemical marker of proliferation in brain tumors. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Polyamines; Ornithine decarboxylase; Glioma; Brain tumor; Cat
Metabolism of the polyamines spermidine and spermine and their precursor putrescine is mainly regulated by changes in the activity of the first key enzyme ornithine decarboxylase (ODC), which catalyzes the decarboxylation of the amino acid ornithine to the diamine putrescine (Pegg, 1986; Morgan, 1987). Activation of polyamine biosynthesis is closely associated with both physiological cell growth, proliferation and regeneration (Canellakis et al., 1979; Heby, 1981; Seiler, 1981) and pathological proliferation processes (Horn et al., 1982; Ja¨nne et al., 1991; Auvinen, 1997). Cell cycle studies indicated that ODC induction is a universal feature of the G1-phase of the cell and is a mandatory * Corresponding author. Present address: Klinik fu¨r Neurochirurgie der Universita¨t zu Ko¨ln, Joseph-Stelzmann-Strasse 9, D-50924 Ko¨ln, Germany. Tel.: +49-221-4784560; fax: +49-221-4786257. E-mail address: [email protected] (G. Ro¨hn).
event for the cells as they progress through G1-and enter S-phase (Russell and Haddox, 1978). ODC responds rapidly to a wide variety of metabolic stimuli with changes in its activity, due to an increased amount of ODC protein (Pegg, 1986). The expression of the enzyme is mainly regulated post-transcriptionally (Persson et al., 1986; Ja¨nne et al., 1991). In the brain, the metabolic rate of polyamines is very low under physiological conditions (Harik and Snyder, 1974; Seiler and Schmidt-Glenewinkel, 1975; Russell and Durie, 1978). Therefore, changes in ODC activity (Raina et al., 1976) and the amount of polyamines may reflect pathological growth processes (Scalabrino and Ferioli, 1981, 1982). In a first series of experiments, we investigated ODC activity and levels of polyamines regionally in rat brains after tumor growth (Ernestus et al., 1993). In the tumor, both, ODC activity and polyamine levels were
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G. Ro¨hn et al. / Neurochemistry International 39 (2001) 135–140
markedly elevated as compared to non-neoplastic brain tissue. However, increase in enzymatic activity occurred exclusively in the tumor, whereas the polyamines also rised throughout the vicinity of the tumor in the ipsilateral hemisphere. These findings suggested that activation of polyamine metabolism, as expressed by an increase of ODC activity, might be a biochemical marker of neoplastic growth in the brain. In the present study, we wanted to investigate the regional distribution of ODC activity and polyamines in more detail. Therefore, we chose the well characterized model of experimental cat brain tumor (Hossmann et al., 1983, 1989; Wechsler et al., 1989; Hoehn-Berlage et al., 1992), which allows a differentiation and correlation of biochemical and histomorphological findings.
1. Experimental procedures
1.1. Animal experiments Experimental brain tumors were produced in three mongrel cats (3.1–4.7 kg body weight) by stereotactic xenotransplantation of cells of the rat glioma clone F98 (courtesy of Professor Wechsler, Du¨ sseldorf, Germany) under sterile conditions. The cats were anaesthetized with ketamine-halothane (5 mg/kg body weight Ketanest®, 3.45 mg i.p. atropine, and 1– 1.5% halothane in a mixture of 70% N2O and 30% O2) and fixed in a stereotactic head holder (LPC, Paris, France). Through a small burr hole (anterior 18 mm, lateral 8 mm, depth 3.5 mm following Horsley-Clark coordinates), 10 ml of the cell suspension (106 cells) were slowly injected into the internal capsule of the left hemisphere. After implantation, the skin wound was sutured and the animals returned to their cage. Twenty days after tumor implantation, the animals were reanaesthetized as described above. The cats were intubated, immobilized (0.5 mg pancuronium bromide i.v.) and artificially ventilated. The left femoral vein and artery were canulated for monitoring arterial blood pressure and blood gases, and for drug application. The rectal temperature probe was connected to a feedbackcontrolled water jacket covering the body of the animal. Body temperature was kept constant at 36.3 91.3°C throughout the experimental period. Blood gases were maintained within the physiological range by appropriate adjustments of ventilation. A skin incision was performed to expose the cranium and the brains were frozen in situ by pouring liquid nitrogen on it (Ponte´ n et al., 1973). Fifteen minutes after starting freezing, cardiac arrest was caused by intravenous injection of saturated potassium chloride. The animals were decapitated and the head placed in liquid nitrogen and stored at − 80°C until further investigation.
A control animal was treated in like manner as described above except the inoculation of tumor cells. 1.2. Histology The frozen brains were cut with a band saw into 1 cm thick coronal slices under intermittent liquid nitrogen irrigation. Bone and surrounding tissue were removed in a glovebox at − 20°C and cryostat sections of 10 mm were taken at the level of the tumor center for histological investigation. Slices were stained using hematoxylin and eosin (HE) to define the tumor area. For representation of peritumoral edema, an immunohistochemical staining of extravasal immunogobulines with the peroxidase–antiperoxidase (PAP)method was carried out (Sternberger et al., 1970; Hossmann et al., 1980; Wechsler et al., 1989). A scanning pattern with 60–80 fields was laid on the cut surface of the brain for the determination of both, the mitotic rate and the corresponding biochemical data. The mitotic index for each field given as mitoses per mm2 was correlated to the level of ODC activity in the same field. Statistical analysis was performed by linear regression analysis (StatView 512+™).
1.3. Tissue sampling and biochemical assays Corresponding to the above mentioned scanning pattern, tissue samples (5–10 mg) were taken from each field, first for the determination of ODC activity. Subsequently, tissue samples for the determination of polyamines were taken from the adjacent section. Quantitative biochemical analysis was performed as described previously (Djuricic et al., 1988; Paschen et al., 1988). In brief, ODC activity was measured by quantifying the release of 14CO2 from D-[1-14C]ornithine (spec. act. 2.06 TBq/mol, NEN-DuPont, Bad Homburg, Germany). Brain samples were homogenized at 4°C with 25 volume (w/v) of Tris/HCl buffer (50 mM, pH 7.2), supplemented with 5 mM dithiothreitol and 0.1 mM EGTA. The assay was carried out in sealed tubes with center wells. The test mixture was composed of tissue homogenate (4 mg tissue), pyridoxal-5-phosphate (54 mM) and [14C]ornithine (0.5 mCi) in a total volume of 130 ml. For determination of polyamine levels, tissue samples were homogenized at −20°C with 50 ml of 0.1 M HCl in methanol and the homogenates were extracted twice with 0.6 M perchloric acid (50–100 ml). The extracts were neutralized with 3 M KOH. After derivatization with o-phthalaldehyd polyamines were separated by means of a reversed-phase HPLC column (Partisil 10 ODS 3) and quantified by fluorescence detection. The quantified data were color-coded to present the distribution of ODC-activity and polyamine levels in the respective section. Low values were colored in blue and green, high values in yellow and red.
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Fig. 1. Correlation of ODC activity, polyamine levels, and histology in experimental cat brain tumors 20 days after stereotactic tumor cell implantation. Figures presented in a vertical row are corresponding to one brain. (A) Tumor with central necrosis (HE); (B) High number of cells and mitoses in the area with highest ODC activity (HE, × 600); (C) Regional distribution of ODC activity; (D) Tumor with little necrosis (HE); (E) Immunohistochemical staining of immunoglobulin extravasation in the peritumoral edema (PAP); (F) Regional distribution of putrescine content; (G) Regional distribution of spermidine content; (H) Regional distribution of spermine content.
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peritumoral edema resulted in a pronounced shift of the midline to the opposite side.
2.2. ODC acti6ity
Fig. 2. Correlation of regional ODC activity and the mitotic rate in experimental cat brain tumors 20 days after stereotactic tumor cell implantation. Linear regression analysis reveals a regression coefficient A =0.383 and a correlation coefficient r = 0.861.
2. Results
2.1. Tumor growth and histology After implantation of the tumor cells, the animals quickly recovered. They developed a fairly large (1–1.5 cm), well defined tumor in the white matter of the left hemisphere (Fig. 1A, D). Neither disabilities in behavior nor any focal neurological deficits during tumor growth could be observed. HE-staining revealed a well demarcated tumor with little peritumoral leuco- and lymphocytic infiltrations indicating the beginning of repellent reactions (Fig. 1A, D). The cell rich tumor was composed of solid and cystic-necrotic areas (Fig. 1A). Microscopically, the tumor cells were mainly uniform and showed a lot of mitoses (Fig. 1B). The mitotic rate widely varied within the tumor with a maximum of 29 mitoses per mm2 (Fig. 2). In summary, histological findings corresponded to a polymorphous, highly malignant anaplastic glioma. Immunohistochemical staining of the extravasation of immunoglobulins showed an extended peritumoral edema with a typical spreading into the white matter (Fig. 1E). The combined mass increase of tumor and
ODC activity of the contralateral hemisphere (0.9– 4.1 nmol/g per hour) was not substantially different to the brain of the healthy control animal (0.9–5.8 nmol/g per hour, Table 1). Within the tumor, areas with highly increased enzyme activity were apparent, whereas measurements of the peritumoral tissue of the ipsilateral hemisphere did not differ from the non-tumoral contralateral hemisphere (Fig. 1C). However, increase of ODC activity was not uniform throughout the whole tumor. ODC activity markedly rised in parallel with increasing mitotic rate, the correlation coefficient of both parameters amounted to r= 0.861 (Fig. 2). Highest ODC values (11.2 nmol/g per hour) could be measured in the medial border zone of the tumor (Fig. 1C). This area also exhibited higher cell density and mitotic rate as compared to the lateral border (Fig. 1B). Thus, the appearance of ODC activity correlated with the amount of proliferating tumor parts. In necrotic parts of the tumor, only low enzyme activity could be detected (0.9–1.5 nmol/g per hour, Fig. 1C)
2.3. Polyamine le6els Regional polyamine levels in the contralateral hemisphere of tumor-bearing cats corresponded to the amount measured in the control animal (Table 1). Putrescine levels were markedly increased in the whole tumor (63–225 nmol/g) as compared to levels of the contralateral hemisphere (12–42 nmol/g, Fig. 1F). However, spermidine (Fig. 1G) and spermine (Fig. 1H) were only slightly increased in some parts of the tumor. In contrast to the distribution of ODC activity, regional differences did not correlate neither with solid or necrotic tumor areas nor with the proliferation of vital parts of the tumor. Furthermore, an increase in putrescine could also be observed in the peritumoral edema (up to 188 nmol/g), whereas spermidine (up to 404 nmol/g) and spermine (up to 234 nmol/g) did not show any changes as compared to the contralateral hemisphere (Table 1).
Table 1 Regional variation of ODC activity and polyamine content in experimental cat brain gliomas 20 days after stereotactic tumor cell implantation and in a healthy control animal without tumor
Control Contralateral Ipsilateral Tumor
ODC (nmol/g per hour)
Putrescine (nmol/g)
Spermidine (nmol/g)
Spermine (nmol/g)
0.9–5.8 0.9–4.1 0.7–4.0 0.5–11.2
4–36 12–42 5–188 63–225
54–456 30–466 11–404 70–841
5–254 8–219 10–234 19–349
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3. Discussion The association of cell proliferation and activation of polyamine synthesis was fundamental for a lot of biochemical studies in experimental and human brain tumors. These investigations revealed that the metabolic activation, as expressed by the activity of ODC, is restricted to the tumor tissue itself (Scalabrino et al., 1982; Scalabrino and Ferioli, 1985; Ernestus et al., 1993) and, furthermore, that the degree of activation depends on the grade of malignancy (Scalabrino et al., 1982; Scalabrino and Ferioli, 1985; Ernestus et al., 1996). Thus, it was concluded that the degree of ODC activation represents a biochemical marker of proliferation in the brain. However, despite the large number of studies not only on brain tumors but also on extraneural neoplasms (Scalabrino and Ferioli, 1981, 1982; Pegg, 1988; Ja¨ nne et al., 1991), no regional differentiation of the degree of metabolic activation within individual tumors was attempted so far. In the present study of cat brain gliomas, activation of polyamine metabolism was investigated for the first time regionally within brain tumors. These experimental tumors are anaplastic gliomas induced by stereotactical xenotransplantation of cloned gliomas cells of the rat. They are characterized, comparable to human glioblastoma multiforme (Kleihues et al., 1993; Davis and Robertson, 1997), by the coincidence of solid tumor parts with a large number of mitoses and necrotic tumor areas (Wechsler et al., 1989). It could be shown that the degree of metabolic activation is not homogeneous but, corresponding to the histomorphologic heterogeneity of these anaplastic gliomas, characterized by locally marked differences. Tumor parts with a high mitotic index corresponded to those areas exhibiting highest ODC activity. Thus, these in situ experiments confirm the results of Heby et al. (1975), who found a close relation between the specific growth fraction of malignant glioma cells and ODC activity in vitro. As already shown in experimental rat brain gliomas (Ernestus et al., 1993), no increase of ODC activity could be observed in the peritumoral brain as compared to the contralateral hemisphere. However, putrescine level was increased, as compared to the contralateral hemisphere, not only in solid and proliferating tumor parts, but also in necrotic areas and in the peritumoral, non-neoplastic brain. These findings confirm the hypothesis that polyamines are synthesized within the tumor cells and spread into the extracellular space by active transport processes (Seiler and Dezeure, 1990; Gilad and Gilad, 1991) as well as by spontaneous or therapeutic tumor regression (Russell, 1983). The differences in the distribution of polyamines within the tumor and peritumoral edema may reflect their different characteristics in uptake, binding and release. Gilad
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and Gilad (1991) could show that specific and highaffinity uptake by rat synaptosomes is unique to putrescine, whereas specific binding is seen only for spermidine and spermine, but not for putrescine. Release of putrescine into the extracellular compartment may disturb the integrity of neurons by overactivating their N-methyl-D-aspartate receptors and enhanced calcium fluxes as it has been shown after ischemia (Koenig et al., 1990; Paschen et al., 1991). On the other hand, polyamine depletion is able to inhibit tumor growth of U-251 human glioblastoma in nude mice (Moulinoux et al., 1991). In conclusion, the correlation of regional mitotic and ODC activity within experimental gliomas underlines the relevance of ODC as a proliferation marker in brain tumors. However, the visualization of metabolic activation is restricted to in situ experiments. Until now, no in vivo method is available for the selective demonstration of ODC activation in brain tumors. Further studies leaving the level of quantitative biochemistry are required both for the in vivo detection of ODC activation by positron emission tomography (PET) and/or magnetic resonance spectroscopy (MRS) and for the elucidation of the role of ODC on the level of molecular biology. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, Grant Pa 266/3-2. The excellent technical assistance of A8 nne Pribliczki, Beate Joschko and Barbara Kokoscha is gratefully acknowledged. References Auvinen, M., 1997. Cell transformation, invasion, and angiogenesis: a regulatory role for ornithine decarboxylase. Journal of the National Cancer Institute 85, 533 – 537. Canellakis, E.S., Viceps-Madore, D., Kyriakidis, D.A., Heller, J.S., 1979. The regulation and function of ornithine decarboxylase and of the polyamines. Current Topics in Cellular Regulation 15, 155 – 202. Davis, L.R., Robertson, D.M. (Eds.), 1997. Textbook of Neuropathology, 3rd ed. Williams and Wilkins, Baltimore. Djuricic, B.M., Paschen, W., Schmidt-Kastner, R., 1988. Polyamines in the brain: HPLC analysis and its application in cerebral ischemia. Iugoslavica Physiologica et Pharmacologica Acta 24, 9 – 17. Ernestus, R.-I., Ro¨ hn, G., Hossmann, K.-A., Paschen, W., 1993. Polyamine metabolism in experimental brain tumors of rat. Journal of Neurochemistry 60, 417 – 422. Ernestus, R.-I., Ro¨ hn, G., Schro¨ der, R., Els, T., Lee, J.-Y., Klug, N., Paschen, W., 1996. Polyamine metabolism in gliomas. Journal of Neuro-Oncology 29, 167 – 174. Gilad, G.M., Gilad, V.H., 1991. Polyamine uptake, binding and release in rat brain. European Journal of Pharmcology 193, 41 – 46.
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Harik, S.I., Snyder, S.H., 1974. Putrescine. Regional distribution in the nervous system of the rat and the cat. Brain Research 66, 328 – 331. Heby, O., 1981. Role of polyamines in the control of cell proliferation and differentiation. Differentiation 19, 1 –20. Heby, O., Marton, L.J., Wilson, C.B., Martinez, H.M., 1975. Polyamine metabolism in a rat brain tumor cell line: its relationship to the growth rate. Journal of Cellular Physiology 86, 511 – 522. Hoehn-Berlage, M., Tolxdorff, T., Bockhorst, K., Okada, Y., Ernestus, R.-I., 1992. In vivo NMR T2 relaxation of experimental brain tumors in the cat: a multiparameter tissue characterization. Magnetic Resonance Imaging 10, 935 – 947. Horn, Y., Beal, S.L., Walach, N., Lubich, W.P., Spigel, L., Marton, L.J., 1982. Further evidence for the use of polyamines as biochemical markers for malignant tumors. Cancer Research 42, 3248 – 3251. Hossmann, K.-A., Blo¨ ink, M., Wilmes, F., Wechsler, W., 1980. Experimental peritumoral edema of the cat brain. Advances in Neurology 28, 323 – 340. Hossmann, K.-A., Hu¨ rter, T., Oschlies, U., 1983. The effect of dexamethasone on serum protein extravasation and edema development in experimental brain tumors of cat. Acta Neuropathologica (Berl.) 60, 223 –231. Hossmann, K.-A., Szymas, J., Seo, K., Assheuer, J., Krajewski, S., 1989. Experimental transplantation gliomas in the adult cat brain. 2. Pathophysiology and magnetic resonance imaging. Acta Neurochirurgica (Wien) 98, 189 –200. Ja¨ nne, J., Alhonen, L., Leinonen, P., 1991. Polyamines: From molecular biology to clinical applications. Annals of Medicine 23, 241 – 259. Kleihues, P., Burger, P.C., Scheithauer, B.W., 1993. Histological Typing of Tumours of the Central Nervous System. Springer, Berlin/Heidelberg/New York. Koenig, H., Goldstone, A.D., Lu, C.Y., Trout, J.J., 1990. Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischemia and recirculation. Stroke 21 (Suppl. III), III-98 – III102. Morgan, D.M.L., 1987. Polyamines. Essays in Biochemistry 23, 82 – 115. Moulinoux, J.-P., Darcel, F., Quemener, V., Havouis, R., Seiler, N., 1991. Inhibition of the growth of U-251 human glioblastoma in nude mice by polyamine deprivation. Anticancer Research 11, 175 – 180. Paschen, W., Ro¨ hn, G., Meese, C.O., Djuricic, B., Schmidt-Kastner, R., 1988. Polyamine metabolism in reversible cerebral ischemia: effect of a-difluoromethylornithine. Brain Research 453, 9 – 16. Paschen, W., Csiba, L., Ro¨ hn, G., Bereczki, D., 1991. Polyamine metabolism in transient focal ischemia of rat brain. Brain Research 566, 354 – 357.
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Pegg, A.E., 1986. Recent advances in the biochemistry of polyamines in eukaryotes. Biochemical Journal 234, 249 – 262. Pegg, A.E., 1988. Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Research 48, 759 – 774. Persson, L., Holm, I., Heby, O., 1986. Translational regulation of ornithine decarboxylase by polyamines. FEBS Letters 205, 175 – 178. Ponte´ n, U., Ratcheson, R.A., Salford, L.G., Siesjo¨ , B.K., 1973. Optimal freezing conditions for cerebral metabolites in rats. Journal of Neurochemistry 21, 1127 – 1138. Raina, A., Pajula, R.L., Eloranta, T., 1976. Distribution of polyamine synthesizing enzymes and methionine adenosyltransferase in rat tissues. FEBS Letters 67, 252 – 256. Russell, D.H., 1983. Clinical relevance of polyamines. Critical Reviews in Clinical Laboratory Sciences 18, 261 – 311. Russell, D.H., Haddox, M.K., 1978. Cyclic AMP-mediated induction of ornithine decarboxylase in normal and neoplastic growth. Advances in Enzyme Regulation 17, 61 – 87. Russell, D.H., Durie, B.G.M., 1978. Polyamines as Biochemical Markers of Normal and Malignant Growth. Raven Press, New York. Scalabrino, G., Ferioli, M.E., 1981. Polyamines in mammalian tumors. Part I. Advances in Cancer Research 35, 151 – 268. Scalabrino, G., Ferioli, M.E., 1982. Polyamines in mammalian tumors. Part II. Advances in Cancer Research 36, 1 – 102. Scalabrino, G., Ferioli, M.E., 1985. Degree of enhancement of polyamine biosynthetic decarboxylase activities in human tumors: a useful new index of degree of malignancy. Cancer Detection and Prevention 8, 11 – 16. Scalabrino, G., Modena, D., Ferioli, M.E., Puerari, M., Luccarelli, G., 1982. Degrees of malignancy in human primary central nervous system tumors: ornithine decarboxylase levels as better indicators than adenosylmethionine decarboxylase levels. Journal of the National Cancer Institute 68, 751 – 754. Seiler, N., 1981. Polyamine metabolism and function in brain. Neurochemistry International 3, 95 – 110. Seiler, N., Dezeure, F., 1990. Polyamine transport in mammalian cells. International Journal of Biochemistry 22, 211 – 218. Seiler, N., Schmidt-Glenewinkel, T., 1975. Regional distribution of putrescine, spermidine and spermine in relation to the distribution of RNA and DNA in the rat nervous system. Journal of Neurochemistry 24, 791 – 795. Sternberger, L.A., Hardy, P.H. Jr, Cuculis, J.J., Meyer, H.G., 1970. The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. Journal of Histochemistry and Cytochemistry 18, 315 – 333. Wechsler, W., Szymas, J., Bilzer, T., Hossmann, K.-A., 1989. Experimental transplantation gliomas in the adult cat brain. Acta Neurochirurgica (Wien) 98, 77 – 89.
Regional distribution of ornithine decarboxylase activity and polyamine levels in experimental cat brain tumors Gabriele Ro¨hn a, Thomas Els b, Karen Hell c, Ralf-Ingo Ernestus a,* b
a Department of Neurosurgery, Uni6ersity of Cologne, Cologne, Germany Department of Experimental Neurology, Max-Planck-Institute for Neurological Research, Cologne, Germany c Department of Pathology, Uni6ersity of Cologne, Cologne, Germany
Received 2 June 2000; received in revised form 3 January 2001; accepted 8 January 2001
Abstract Biosynthesis of the polyamines putrescine, spermidine, and spermine, and activation of the first key enzyme ornithine decarboxylase (ODC) are closely associated with cellular proliferation. In the present study, the distribution of ODC activity and polyamine levels was investigated for the first time regionally in experimental brain tumors of the cat. Brain tumors were produced by stereotactic xenotransplantation of rat glioma cells. Twenty days after implantation, the brains were frozen in situ, cut into slices, and cryostat sections and tissue samples were taken to determine ODC activity and polyamine levels biochemically. The quantified data were color-coded to present the regional distribution of ODC activity and polyamine levels in the respective section. ODC activity significantly increased in some areas within the tumor, whereas peritumoral tissue showed no difference to the non-tumoral, contralateral hemisphere. This increase turned out in parallel to a high number of mitoses in the same tumor parts (r =0.861). Putrescine levels increased both, in the whole tumor and in the peritumoral edema. Regional differences in putrescine content did not correlate with solid and proliferative parts of the tumor. Spermidine and spermine levels were only slightly increased in some parts of the tumor. Thus, these experiments show the close correlation of a high mitotic rate and activation of ODC within experimental gliomas and underline the relevance of ODC as a biochemical marker of proliferation in brain tumors. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Polyamines; Ornithine decarboxylase; Glioma; Brain tumor; Cat
Metabolism of the polyamines spermidine and spermine and their precursor putrescine is mainly regulated by changes in the activity of the first key enzyme ornithine decarboxylase (ODC), which catalyzes the decarboxylation of the amino acid ornithine to the diamine putrescine (Pegg, 1986; Morgan, 1987). Activation of polyamine biosynthesis is closely associated with both physiological cell growth, proliferation and regeneration (Canellakis et al., 1979; Heby, 1981; Seiler, 1981) and pathological proliferation processes (Horn et al., 1982; Ja¨nne et al., 1991; Auvinen, 1997). Cell cycle studies indicated that ODC induction is a universal feature of the G1-phase of the cell and is a mandatory * Corresponding author. Present address: Klinik fu¨r Neurochirurgie der Universita¨t zu Ko¨ln, Joseph-Stelzmann-Strasse 9, D-50924 Ko¨ln, Germany. Tel.: +49-221-4784560; fax: +49-221-4786257. E-mail address: [email protected] (G. Ro¨hn).
event for the cells as they progress through G1-and enter S-phase (Russell and Haddox, 1978). ODC responds rapidly to a wide variety of metabolic stimuli with changes in its activity, due to an increased amount of ODC protein (Pegg, 1986). The expression of the enzyme is mainly regulated post-transcriptionally (Persson et al., 1986; Ja¨nne et al., 1991). In the brain, the metabolic rate of polyamines is very low under physiological conditions (Harik and Snyder, 1974; Seiler and Schmidt-Glenewinkel, 1975; Russell and Durie, 1978). Therefore, changes in ODC activity (Raina et al., 1976) and the amount of polyamines may reflect pathological growth processes (Scalabrino and Ferioli, 1981, 1982). In a first series of experiments, we investigated ODC activity and levels of polyamines regionally in rat brains after tumor growth (Ernestus et al., 1993). In the tumor, both, ODC activity and polyamine levels were
0197-0186/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0197-0186(01)00011-0
136
G. Ro¨hn et al. / Neurochemistry International 39 (2001) 135–140
markedly elevated as compared to non-neoplastic brain tissue. However, increase in enzymatic activity occurred exclusively in the tumor, whereas the polyamines also rised throughout the vicinity of the tumor in the ipsilateral hemisphere. These findings suggested that activation of polyamine metabolism, as expressed by an increase of ODC activity, might be a biochemical marker of neoplastic growth in the brain. In the present study, we wanted to investigate the regional distribution of ODC activity and polyamines in more detail. Therefore, we chose the well characterized model of experimental cat brain tumor (Hossmann et al., 1983, 1989; Wechsler et al., 1989; Hoehn-Berlage et al., 1992), which allows a differentiation and correlation of biochemical and histomorphological findings.
1. Experimental procedures
1.1. Animal experiments Experimental brain tumors were produced in three mongrel cats (3.1–4.7 kg body weight) by stereotactic xenotransplantation of cells of the rat glioma clone F98 (courtesy of Professor Wechsler, Du¨ sseldorf, Germany) under sterile conditions. The cats were anaesthetized with ketamine-halothane (5 mg/kg body weight Ketanest®, 3.45 mg i.p. atropine, and 1– 1.5% halothane in a mixture of 70% N2O and 30% O2) and fixed in a stereotactic head holder (LPC, Paris, France). Through a small burr hole (anterior 18 mm, lateral 8 mm, depth 3.5 mm following Horsley-Clark coordinates), 10 ml of the cell suspension (106 cells) were slowly injected into the internal capsule of the left hemisphere. After implantation, the skin wound was sutured and the animals returned to their cage. Twenty days after tumor implantation, the animals were reanaesthetized as described above. The cats were intubated, immobilized (0.5 mg pancuronium bromide i.v.) and artificially ventilated. The left femoral vein and artery were canulated for monitoring arterial blood pressure and blood gases, and for drug application. The rectal temperature probe was connected to a feedbackcontrolled water jacket covering the body of the animal. Body temperature was kept constant at 36.3 91.3°C throughout the experimental period. Blood gases were maintained within the physiological range by appropriate adjustments of ventilation. A skin incision was performed to expose the cranium and the brains were frozen in situ by pouring liquid nitrogen on it (Ponte´ n et al., 1973). Fifteen minutes after starting freezing, cardiac arrest was caused by intravenous injection of saturated potassium chloride. The animals were decapitated and the head placed in liquid nitrogen and stored at − 80°C until further investigation.
A control animal was treated in like manner as described above except the inoculation of tumor cells. 1.2. Histology The frozen brains were cut with a band saw into 1 cm thick coronal slices under intermittent liquid nitrogen irrigation. Bone and surrounding tissue were removed in a glovebox at − 20°C and cryostat sections of 10 mm were taken at the level of the tumor center for histological investigation. Slices were stained using hematoxylin and eosin (HE) to define the tumor area. For representation of peritumoral edema, an immunohistochemical staining of extravasal immunogobulines with the peroxidase–antiperoxidase (PAP)method was carried out (Sternberger et al., 1970; Hossmann et al., 1980; Wechsler et al., 1989). A scanning pattern with 60–80 fields was laid on the cut surface of the brain for the determination of both, the mitotic rate and the corresponding biochemical data. The mitotic index for each field given as mitoses per mm2 was correlated to the level of ODC activity in the same field. Statistical analysis was performed by linear regression analysis (StatView 512+™).
1.3. Tissue sampling and biochemical assays Corresponding to the above mentioned scanning pattern, tissue samples (5–10 mg) were taken from each field, first for the determination of ODC activity. Subsequently, tissue samples for the determination of polyamines were taken from the adjacent section. Quantitative biochemical analysis was performed as described previously (Djuricic et al., 1988; Paschen et al., 1988). In brief, ODC activity was measured by quantifying the release of 14CO2 from D-[1-14C]ornithine (spec. act. 2.06 TBq/mol, NEN-DuPont, Bad Homburg, Germany). Brain samples were homogenized at 4°C with 25 volume (w/v) of Tris/HCl buffer (50 mM, pH 7.2), supplemented with 5 mM dithiothreitol and 0.1 mM EGTA. The assay was carried out in sealed tubes with center wells. The test mixture was composed of tissue homogenate (4 mg tissue), pyridoxal-5-phosphate (54 mM) and [14C]ornithine (0.5 mCi) in a total volume of 130 ml. For determination of polyamine levels, tissue samples were homogenized at −20°C with 50 ml of 0.1 M HCl in methanol and the homogenates were extracted twice with 0.6 M perchloric acid (50–100 ml). The extracts were neutralized with 3 M KOH. After derivatization with o-phthalaldehyd polyamines were separated by means of a reversed-phase HPLC column (Partisil 10 ODS 3) and quantified by fluorescence detection. The quantified data were color-coded to present the distribution of ODC-activity and polyamine levels in the respective section. Low values were colored in blue and green, high values in yellow and red.
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Fig. 1. Correlation of ODC activity, polyamine levels, and histology in experimental cat brain tumors 20 days after stereotactic tumor cell implantation. Figures presented in a vertical row are corresponding to one brain. (A) Tumor with central necrosis (HE); (B) High number of cells and mitoses in the area with highest ODC activity (HE, × 600); (C) Regional distribution of ODC activity; (D) Tumor with little necrosis (HE); (E) Immunohistochemical staining of immunoglobulin extravasation in the peritumoral edema (PAP); (F) Regional distribution of putrescine content; (G) Regional distribution of spermidine content; (H) Regional distribution of spermine content.
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peritumoral edema resulted in a pronounced shift of the midline to the opposite side.
2.2. ODC acti6ity
Fig. 2. Correlation of regional ODC activity and the mitotic rate in experimental cat brain tumors 20 days after stereotactic tumor cell implantation. Linear regression analysis reveals a regression coefficient A =0.383 and a correlation coefficient r = 0.861.
2. Results
2.1. Tumor growth and histology After implantation of the tumor cells, the animals quickly recovered. They developed a fairly large (1–1.5 cm), well defined tumor in the white matter of the left hemisphere (Fig. 1A, D). Neither disabilities in behavior nor any focal neurological deficits during tumor growth could be observed. HE-staining revealed a well demarcated tumor with little peritumoral leuco- and lymphocytic infiltrations indicating the beginning of repellent reactions (Fig. 1A, D). The cell rich tumor was composed of solid and cystic-necrotic areas (Fig. 1A). Microscopically, the tumor cells were mainly uniform and showed a lot of mitoses (Fig. 1B). The mitotic rate widely varied within the tumor with a maximum of 29 mitoses per mm2 (Fig. 2). In summary, histological findings corresponded to a polymorphous, highly malignant anaplastic glioma. Immunohistochemical staining of the extravasation of immunoglobulins showed an extended peritumoral edema with a typical spreading into the white matter (Fig. 1E). The combined mass increase of tumor and
ODC activity of the contralateral hemisphere (0.9– 4.1 nmol/g per hour) was not substantially different to the brain of the healthy control animal (0.9–5.8 nmol/g per hour, Table 1). Within the tumor, areas with highly increased enzyme activity were apparent, whereas measurements of the peritumoral tissue of the ipsilateral hemisphere did not differ from the non-tumoral contralateral hemisphere (Fig. 1C). However, increase of ODC activity was not uniform throughout the whole tumor. ODC activity markedly rised in parallel with increasing mitotic rate, the correlation coefficient of both parameters amounted to r= 0.861 (Fig. 2). Highest ODC values (11.2 nmol/g per hour) could be measured in the medial border zone of the tumor (Fig. 1C). This area also exhibited higher cell density and mitotic rate as compared to the lateral border (Fig. 1B). Thus, the appearance of ODC activity correlated with the amount of proliferating tumor parts. In necrotic parts of the tumor, only low enzyme activity could be detected (0.9–1.5 nmol/g per hour, Fig. 1C)
2.3. Polyamine le6els Regional polyamine levels in the contralateral hemisphere of tumor-bearing cats corresponded to the amount measured in the control animal (Table 1). Putrescine levels were markedly increased in the whole tumor (63–225 nmol/g) as compared to levels of the contralateral hemisphere (12–42 nmol/g, Fig. 1F). However, spermidine (Fig. 1G) and spermine (Fig. 1H) were only slightly increased in some parts of the tumor. In contrast to the distribution of ODC activity, regional differences did not correlate neither with solid or necrotic tumor areas nor with the proliferation of vital parts of the tumor. Furthermore, an increase in putrescine could also be observed in the peritumoral edema (up to 188 nmol/g), whereas spermidine (up to 404 nmol/g) and spermine (up to 234 nmol/g) did not show any changes as compared to the contralateral hemisphere (Table 1).
Table 1 Regional variation of ODC activity and polyamine content in experimental cat brain gliomas 20 days after stereotactic tumor cell implantation and in a healthy control animal without tumor
Control Contralateral Ipsilateral Tumor
ODC (nmol/g per hour)
Putrescine (nmol/g)
Spermidine (nmol/g)
Spermine (nmol/g)
0.9–5.8 0.9–4.1 0.7–4.0 0.5–11.2
4–36 12–42 5–188 63–225
54–456 30–466 11–404 70–841
5–254 8–219 10–234 19–349
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3. Discussion The association of cell proliferation and activation of polyamine synthesis was fundamental for a lot of biochemical studies in experimental and human brain tumors. These investigations revealed that the metabolic activation, as expressed by the activity of ODC, is restricted to the tumor tissue itself (Scalabrino et al., 1982; Scalabrino and Ferioli, 1985; Ernestus et al., 1993) and, furthermore, that the degree of activation depends on the grade of malignancy (Scalabrino et al., 1982; Scalabrino and Ferioli, 1985; Ernestus et al., 1996). Thus, it was concluded that the degree of ODC activation represents a biochemical marker of proliferation in the brain. However, despite the large number of studies not only on brain tumors but also on extraneural neoplasms (Scalabrino and Ferioli, 1981, 1982; Pegg, 1988; Ja¨ nne et al., 1991), no regional differentiation of the degree of metabolic activation within individual tumors was attempted so far. In the present study of cat brain gliomas, activation of polyamine metabolism was investigated for the first time regionally within brain tumors. These experimental tumors are anaplastic gliomas induced by stereotactical xenotransplantation of cloned gliomas cells of the rat. They are characterized, comparable to human glioblastoma multiforme (Kleihues et al., 1993; Davis and Robertson, 1997), by the coincidence of solid tumor parts with a large number of mitoses and necrotic tumor areas (Wechsler et al., 1989). It could be shown that the degree of metabolic activation is not homogeneous but, corresponding to the histomorphologic heterogeneity of these anaplastic gliomas, characterized by locally marked differences. Tumor parts with a high mitotic index corresponded to those areas exhibiting highest ODC activity. Thus, these in situ experiments confirm the results of Heby et al. (1975), who found a close relation between the specific growth fraction of malignant glioma cells and ODC activity in vitro. As already shown in experimental rat brain gliomas (Ernestus et al., 1993), no increase of ODC activity could be observed in the peritumoral brain as compared to the contralateral hemisphere. However, putrescine level was increased, as compared to the contralateral hemisphere, not only in solid and proliferating tumor parts, but also in necrotic areas and in the peritumoral, non-neoplastic brain. These findings confirm the hypothesis that polyamines are synthesized within the tumor cells and spread into the extracellular space by active transport processes (Seiler and Dezeure, 1990; Gilad and Gilad, 1991) as well as by spontaneous or therapeutic tumor regression (Russell, 1983). The differences in the distribution of polyamines within the tumor and peritumoral edema may reflect their different characteristics in uptake, binding and release. Gilad
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and Gilad (1991) could show that specific and highaffinity uptake by rat synaptosomes is unique to putrescine, whereas specific binding is seen only for spermidine and spermine, but not for putrescine. Release of putrescine into the extracellular compartment may disturb the integrity of neurons by overactivating their N-methyl-D-aspartate receptors and enhanced calcium fluxes as it has been shown after ischemia (Koenig et al., 1990; Paschen et al., 1991). On the other hand, polyamine depletion is able to inhibit tumor growth of U-251 human glioblastoma in nude mice (Moulinoux et al., 1991). In conclusion, the correlation of regional mitotic and ODC activity within experimental gliomas underlines the relevance of ODC as a proliferation marker in brain tumors. However, the visualization of metabolic activation is restricted to in situ experiments. Until now, no in vivo method is available for the selective demonstration of ODC activation in brain tumors. Further studies leaving the level of quantitative biochemistry are required both for the in vivo detection of ODC activation by positron emission tomography (PET) and/or magnetic resonance spectroscopy (MRS) and for the elucidation of the role of ODC on the level of molecular biology. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, Grant Pa 266/3-2. The excellent technical assistance of A8 nne Pribliczki, Beate Joschko and Barbara Kokoscha is gratefully acknowledged. References Auvinen, M., 1997. Cell transformation, invasion, and angiogenesis: a regulatory role for ornithine decarboxylase. Journal of the National Cancer Institute 85, 533 – 537. Canellakis, E.S., Viceps-Madore, D., Kyriakidis, D.A., Heller, J.S., 1979. The regulation and function of ornithine decarboxylase and of the polyamines. Current Topics in Cellular Regulation 15, 155 – 202. Davis, L.R., Robertson, D.M. (Eds.), 1997. Textbook of Neuropathology, 3rd ed. Williams and Wilkins, Baltimore. Djuricic, B.M., Paschen, W., Schmidt-Kastner, R., 1988. Polyamines in the brain: HPLC analysis and its application in cerebral ischemia. Iugoslavica Physiologica et Pharmacologica Acta 24, 9 – 17. Ernestus, R.-I., Ro¨ hn, G., Hossmann, K.-A., Paschen, W., 1993. Polyamine metabolism in experimental brain tumors of rat. Journal of Neurochemistry 60, 417 – 422. Ernestus, R.-I., Ro¨ hn, G., Schro¨ der, R., Els, T., Lee, J.-Y., Klug, N., Paschen, W., 1996. Polyamine metabolism in gliomas. Journal of Neuro-Oncology 29, 167 – 174. Gilad, G.M., Gilad, V.H., 1991. Polyamine uptake, binding and release in rat brain. European Journal of Pharmcology 193, 41 – 46.
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