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Isolation and characterization of human breast cancer cells overexpressing S-adenosylmethionine decarboxylase
ELSEVIER
CancerLetters95 (1995)23-28
CANCER LETTERS
Isolation and characterization of human breast cancer cells overexpressing S-adenosylmethionine decarboxylase Andrea Man&*, Betty Badgera,Rhea Grovea,SusanKunselmanb,Laurence Demewc aDepartment of Medicine, Pennsylvania State University School of Medicine, 500 University Drive, P. 0. Box 850, Hershey, PA 17033-0850, USA bCenrerfor Biostatistics and Epidemiology, Pennsylvania State University School ofMedicine. 500 University Drive, P.O. Box 850, Hershey, PA 17033-0850, USA ‘Department qf Pathology, Pennsylvania State University School qf Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17033-0850, USA
Received10May 1995;accepted24May 1995
Abstract
We report the first successful isolation and initial characterization of S-adenosylmethioninedecarboxylase (SAMDC)overexpressingcells using a transfection approach.Stably transfectedMCF-7 breastcancercells overproducing SAMDC (-5fold) manifested reduced omithine decarboxylase while levels of N’-spermidinelspermine acetyltransferasewere variably increased.Analysis of cellular polyamine profile showed that spertnine was selectively increased(-80%), while spermidine and putrescine levels were reduced (-48% and -15% of control, respectively). Since SAMDC-overexpressing clones exhibited increasedclonogenicity in soft agar, our data suggestthat spermine may be selectively involved in conferring a more invasive phenotype to breastcancercells. Keywords: Human breast cancercells; S-Adenosylmethioninedecarboxylase;Polyamines; Soft agar clonogenicity; Proliferative activity
1. Introduction
Polyamines (putrescine, spermidine, and spermine) and their biosynthetic enzymes play an important role in carcinogenesis and neoplastic cell proliferation [I]. Data from our and other laboratories have * Correspondingauthor,Division of Endocrinology,Diabetes and Metabolism,PO Box 850, MS. HersheyMedical Center, Hershev.PA 17033.USA. Tel.: +l 717 5318395;Fax: +l 717 5315726.
demonstrated a critical role for polyamines in breast cancer growth [2]. Furthermore, in hormone-responsive mammary tumors, polyamines appear to be distal effecters of endocrine regulated cell proliferation
PI. Among the critical enzymes controlling the polyamine metabolic pathway, ODC has been the major target for investigation. The availability of several ODC over-producing cell lines resulting from either chronic DEMO exposure [3] or gene transfection [4] has been useful in analyzing the biochemical and
0304-3835/95/$09.500 1995ElsevierScienceIrelandLtd. All rightsreserved SSDI 0304-3835(95)03860-Y
24
A. Manni et al. I Cancer Letters 95 (I 9!?5) 23-28
biological properties of this enzyme. SAMDC, on the other hand, has been implicated to play a predominant role in tumor growth by promoting the formation of the more distal polyamines spermidine and spermine. As a result of this recognition, a recent impetus has been placed on the development of potent SAMDC inhibitors which have been shown to have marked anti-proliferative effects in numerous tumor models [5] including breast cancer [6]. However, in marked contrast to ODC over-producing cell lines, there is a paucity of SAMDC overexpressing cells which can be used to define the biological importance of this enzyme. To the best of our knowledge, there have been only two recent reports of SAMDC over-producing cells generated through selective pressure with an enzyme inhibitor [7,8]. To date, however, no stable overexpression of SAMDC by transfection has been achieved in any experimental system. In this paper, we report for the first time the successful isolation as well as initial characterization of stably transfected breast cancer cells overexpressing SAMDC. 2. Materials and methods 2. I. Cell lines and culture conditions Wild-type MCF-7 breast cancer cells (kindly provided by Dr. V.C. Jordan, University of Wisconsin, Madison, WI) and the transfected clones were propagated in 752 cm flasks in Richter’s improved minimal essential medium (IMEM) (Grand Island Biological, Grand Island, NY) containing phenol red and 5% FBS in a humidified atmosphere of 95% air, 5% CO, at 37°C. In selected experiments (see below), the culture conditions were stepped down to phenol red free IMEM containing dextran-coated charcoal stripped 5% FBS or to serum-free media. 2.2. Plasmid and transfection technique A 1.8 kb human SAMDC cDNA subcloned into an SV40-driven pCD expression vector [9] was kindly provided by Dr. A.E. Pegg (M.S. Hershey Medical Center, Hershey, PA). Transfection of MCF7 cells with pCD SAMDC and an RSV-neo cDNA (ratio 5:l) was carried out using a modified calcium phosphate precipitation technique as previously described by us [4]. Following G-418 selection, we isolated and expanded three control (pCD-3, -5, and
-6) and three SAMDC overexpressing clones (SAM1, -2, and -3). 2.3. Biochemical determinations pCD and pCDSAMDC-transfected clones were grown to near confluency in regular growth medium. For determination of enzymatic activities, the cells were washed three times with ice-cold PBS and then resuspended in buffer containing 50 mM Tris, 2.5 mM DTT, 0.1 mM EDTA (pH 7.5) and stored at -70°C until use. At the time of the assays, the cells were frozen and thawed twice. The cell lysates were centrifuged at 13 600 X g for 20 min. Activities of ODC, SAMDC and SSAT were determined in the cytosolic fraction according to standard techniques in routine use in our laboratory [4,10,11]. For measurement of polyamines, the cells were resuspended in 0.2 N perchloric acid and kept at 4°C overnight. The suspension was then centrifuged at 800 X g for 15 min and supernatant stored until the time of the assay. Polyamines were determined by HPLC with fluorometry as previously described [4]. 2.4. Riological studies Control and SAMDC overexpressing MCF-7 cells were compared with regard to anchorage dependent and independent growth. Plating in soft agar was done as previously published by us [ 121. Cells were plated in triplicate at a density of 1 X lo4 cells/ 35 mm dish. The number of colonies (aggregates > 50 cells) was scored after 20 days. Basal proliferative activity in liquid culture was assessedby plating cells in duplicate at a density of 6 X lo4 cells/35 mm dish in regular growth medium. Twenty-four hours later, the medium was replaced with either the same or phenol red free IMEM containing DCC-treated 5% FBS or serum-free IMEM supplemented with 5 mM glutamine, 0.2 mg% transferrin, 0.1 mg% fibronectin, 0.4 g% fraction V BSA, and 20 mM HEPES buffer. Duplicate dishes per experimental group were harvested on days 2, 4, and 6 and the number of cells was counted using a Colter Counter. 2.5. Statistical methods Analysis of variance (ANOVA) models were used to compare control and SAMDC transfected clones for enzymatic activities and polyamine levels (Table 1). Transformations of enzyme/polyamine levels
A. Munni et ~11.I Cuncer Letters 95 (I 995) 23-28
25
control). ODC activity was markedly suppressed to levels -20% of those detected in control cells. SSAT levels were found to be consistently higher in SAMDC overexpressing breast cancer cells. However, the magnitude of the increase was highly variable from experiment to experiment (see footnote to Table 1). Analysis of cellular polyamine profiles revealed a selective accumulation of spermine (-80% increase over control) while both putrescine and spermidine contents were decreased (-15% and -48% of control, respectively) (Table I). The increase, in spermine probably accounts both for the suppression of ODC and stimulation of SSAT activities. Spermidine and putrescine formed through the back conversion pathway controlled by SSAT are likely to be metabolically converted back to spermine by the increased SAMDC activity. Analysis of the biological properties of our clones revealed that SAMDC overexpressing MCF-7 cells formed more colonies in soft agar (Table 2). This growth advantage was manifested by all three clones and was highly significant in two experiments (experiments 2 and 3) while in experiment 1, only SAM-I exhibited increased clonogenicity. The influence of SAMDC overexpression on an-
were necessary in several cases to meet model assumptions of homogeneous variance and normality. All models included fixed effects for transfection type (vector only versus SAMDC) and experiment and the interaction between the two variables, where significant. For the soft agar experiments reported in Table 2, an ANOVA model was used, taking the square root of the colony number as the response to meet model assumptions. The model included fixed effects for transfection type, experiment and their interaction. Anchorage dependent growth was assessed by linear models which included harvest day, experiment and transfection type. A log transformation was performed on the cell counts to stabilize variance. Second order polynomials were used to model growth over time. All P values correspond to appropriate F-tests. All models were fit using the current version of the SAS statistical software package. 3. Results As can be seen in Table I, significant alterations in the polyamine metabolic pathway resulted from induction of SAMDC overexpression (-5-fold over
Table I Cellular levels of enzymatic activities and polyamines in control (pCD-3, 5, 6) and SAMDC-transfected cmcer cells” Clone
SAMDC (nmol/mg)
ODC (nmol/mg)
SSAT (pmol/mg)
pCD-3
0. I6 2 0.09
I .75 2 0.73
12.94 + 2.9
pCD-5
(10) 0.21 + 0.10
(3) 1.26kO.18
(3) 1.42 + I .6
(12)
(3) 2.59 (3) 0.41 (4) 0.42 (4) 0.33 (4)
(2)
pCD-6 SAM-1
0.17 + 0.07 (11) 0.85 ~0.18
(12) SAM-2 SAM-3
0.87 + 0.22 (11) 0.73 -co.18 (10)
r 1.31 rt 0.23 k 0.20 + 0.17
13.17 + (3) 22.21 * (4) 24.71 + (4) 24.55 t (4)
3.4 7.3 17.2 16.9
(SAM-l,
-2, -3) MCF-7 breast
Polyamines (nmol/mg) F’utrescine
Spermidine
Spermine
4.06 (4) 3.99 (4) 4.77 (4) 0.57 (4) II.62 i4) 0.77 ((4)
14.71 -t- 1.26 (4) 14.18 f 0.87 (4) 14.95 t 1.41 (4) 6.36 r 0.68 (4) 7.54 + 2.98 (4) 7.34 * I .04 (4)
I I .06 + 0.52 (4) 9.31 c 0.4s (4) IO.21 + 1.29 (4) 16.81 2 2.31 (4) 18.76 + 2.39 (4) 19.98 + 2.68 (4)
f 0.4.5 + 0.53 r0.14 * 0.30 + 0.48 + 0.3 1
a Data represent means k SD. Number of determinations is indicated in parentheses. The differences in levels of ODC and SAMDC activities and individual polyamines between control and SAMDC transfected clones were statistically significant (P < 0.0001). The increase in SSAT activity in SAMDC overexpressing cells was variable, being significant in one experiment (P= 0.002) but only marginal in another (P = 0.14).
26
A. Manni et al. I Cancer Letters 95 (1995) 23-28
Table 2 Anchorage independent growth of control and SAMDCoverexpressing MCF-7 breast cancer cellsa Clones
pCD-3 pCD-5 pCD-6 SAM-l SAM-2 SAM-3
Colony number Expt. 1
Expt. 2
Expt. 3
ND 278+ 18 203i7 404*10 270 k 35 192+28
118-+41 135+9 170 k 22 320 + 58 383 f 10 420 f 26
117+20 105 rt 16 155 + 18 241 + 14 201 k 11 259 f 27
12.8
12.6
c 8 1E e z s
A. /
.*-
12.4 -
12.2
-
2
3
a Data represent meansk SD of triplicate dishes/experimental condition. ND, not done. The differences in colony number between control and SAMDC-overexpressing cells were significant in Expts. 2 and 3 (P < 0.0001) but not in Expt. 1 (P = 0.15).
chorage-dependent growth was complex and depended upon the culture conditions. In the presence of regular growth medium, no major differences in proliferative activity were observed between control and SAMDC overexpressing cells (data not shown). When tested under steroid-depleted conditions, SAMDC overproducing cells grew at a faster rate than control (P = 0.04) (Fig. 1A). On the other hand, cell loss, which uniformly occurred under serum-free media conditions, was also significantly enhanced (P = 0.003) by SAMDC overexpression (Fig. 1B). 4. Discussion SAMDC is an enzyme of major importance in polyamine metabolism. It has been proposed that instead of ODC, SAMDC is the rate-limiting enzyme in the formation of spermidine and spermine [3], the polyamines most critically involved in cell proliferation. Hence, evaluation of the biochemical and biological function of this enzyme is of fundamental importance for improving our understanding of polyamine involvement in normal and neoplastic cell growth and for identifying new targets for anti-tumor therapy. The paucity of SAMDC overexpressing experimental systems has limited the investigation of the biochemistry and biology of this enzyme. Such experimental tools would be likely to be useful as shown by the well-proven value of numerous ODC overproducing cells for the study of ODC regulation and function [3,41.
4
6
5
Day
c 3 1c =I 8
10.0-
!! 9.5
-
l .
L 2
-.
l .
l .
3
Day 4
5
lo 6
Fig. 1. Anchorage dependentgrowth of control () (pCD-3, -5, -6) and SAMDC overexpressing(------) (SAM-l, -2, -3) MCF7 breast cancer cells cultured in either DCC-stripped serum contaming medium (A) or serum free medium (B) as specified in Section 2. The data represent the mean log (cell number) from 3 separateexperiments each done in duplicate, In every experiment, all 6 clones were tested.
We report here the first successful generation of SAMDC overexpressing cells using a transfection approach. This provided us with the opportunity to analyze the biochemical changes in the polyamine pathway produced by SAMDC overexpression. As might be anticipated on the basis of the well-known compensatory rise in ODC occurring in response to SAMDC inhibition [5], we observed a significant decrease in ODC activity in our SAMDC overproducing cells. Contrary to our results and surprisingly, ODC activity was found to be either slightly increased or unchanged in mouse FM3A [7] and CHO cells [8] overexpressing SAMDC following chronic
A. Manni et al. I Cancer Letters 95 (1995) 23-28
exposure to enzyme inhibitors. SSAT was found to be increased in our transfected clones, although the magnitude of this increase was variable from experiment to experiment. This finding is in agreement with the well known induction of this enzyme by polyamines [ 131.In our cells, we observed indeed a selective accumulation of spermine which we believe was responsible for the stimulation of SSAT activity. This enzyme, however, did not appear to be increased in SAMDC overexpressing CHO [8] cells and its activity was not reported in mouse FM3A cells [7]. Therefore there appear to be significant differences with regard to the changes in ODC and possibly SSAT activities induced by SAMDC overexpression between our MCF-7 cell clones and the FM3A and CHO cells. Whether these differences are due to the different approach employed to induce SAMDC overexpression (i.e. transfection versus selective pressure with an enzyme inhibitor) or reflect diversity in the intrinsic biologic properties of the cells remain to be determined. The selective increase in cellular spermine associated with a concomitant decrease in putrescine and spermidine contents is consistent with the distal activation of the polyamine pathway induced by SAMDC overexpression which leads to the accumulation of the final product spermine. A similar polyamine profile was also observed in SAMDC overproducing FM3A and CHO cells L',81. Our long-standing interest in polyamines focuses on their role in breast cancer biology. Since polyamines are critically involved in breast cancer cell proliferation and appear to be distal effecters of hormonally stimulated growth [2], we hypothesize that constitutive activation of the polyamine pathway may provide breast cancer cells with a growth advantage and possibly contribute to tumor progression. To test this hypothesis, we used a transfection approach to induce overexpression of key polyamine biosynthetic enzymes in breast cancer cells [4,14]. In previous experiments testing the role of ODC, we observed that ODC overexpressing breast cancer cells manifested a modest growth advantage in liquid culture and reduced sensitivity to estrogens[4,14]. The latter finding suggestsa role of polyamines in the transition of breast cancer to hormone independence. Clonogenicity in soft agar, however, was markedly reduced as a result of ODC overexpression [4]. In marked
21
contrast, as shown here, SAMDC overproducing breast cancer cells exhibited a significant growth advantagein soft agar. This difference probably reflects the different polyamine profiles induced by ODC versus SAMDC overexpression. While ODC overexpressing clones were characterized by selective accumulation of putrescine [4,14], SAMDC overproducing breastcancer cells manifested a selective increasein spermine. Since clonogenicity in soft agar correlates with tumor aggressiveness,these data suggestthe spermine may be selectively involved in conferring a more invasive phenotype to breast cancer cells. The influence of SAMDC overexpression and hence spermine accumulation on anchoragedependent growth was complex and depended upon the culture conditions. When tested in steroiddepletedmedia, SAMDC overexpressingclones grew faster than control cells. On the other hand, cell loss observed in all groups in serum-free media, also occurred at a faster rate in SAMDC transfectedMCF-7 cells. Additional studies will be required to define the mechanismssubservingthesecellular events. Acknowledgments This work is supportedby grant 5 PO1 CA4001 l10 from the National Cancer Institute. References ill Pegg, A.E. (1988) Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res.,48,159-114. PI Manni, A. (1994) The role of polyamines in the hormonal control of breast cancer cell proliferation. In: Mammary Tumorigenesis and Malignant Progression, pp. 209-225. Editors: R. Dickson and M. Lippman. Kluwer Academic, Nonvell, MA. [31 Pegg, A.E., Secrist III, J.A. and Madhubala, R. (1988) Properties of L1210 cells resistant to a-difluoromethylomithine. CancerRes., 48,267&2682. [41 Manni, A., Wechter, R., Wei, L., Heitjan, D. and Demers, L. (1995) Phenotypic features of breast cancer cells overexpressing omithine-decarboxylase. J. Cell. Physiol., 163, 129-136. PI Regenass,U., Mett, H., Stanek, J., Mueller, M., Kramer, D. and Porter, C.W. (1994) CGP 48664, a new Sadenosylmethionine decarboxylase inhibitor with broad spectrum antiproliferative and antitumor activity. Cancer Res.,54.3210-3217.
28
A. Munni et al. I Cuncer Letters 95 (1995) 23-28
[6] Manni, A., Badger, B., Wechter, R. and Demers, L. (1995) Biochemical and growth modulatory effects of the new Sadenosylmethionine decarboxylase inhibitor CGP 48664 in breast cancer cells in culture. Submitted to the 86th Annual Meeting of the American Association for Cancer Research, Toronto, Canada. [7] Suzuki, T., Sadakata, Y., Kashiwagi, K., Hoshino, K., Kakinuma, Y., Shirahata, A. and Igarashi, K. (1993) Overproduction of S-adenosylmethionine decarboxylase in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A cells. Eur. J. Biochem., 215,247-253. [S] Kramer, D., Mett, H., Evans, A., Regenass, U., Diegelman, P. and Porter, C.W. (1995) Stable amplification of the Sadenosylmethionine decarboxylase gene in Chinese hamster ovary cells. J. Biol. Chem., 270,2124-2132. [9] Pajunen, A., Crozat, A., Janne, O.A., lhalainen, R., Laitinen, P.H., Stanley, B., Madhubala. R. and Pegg, A.E. (1988) and regulation of mammalian SStructure adenosylmethionine decarboxylase. J. Biol. Chem., 263, 17040-17049.
1101Pegg, A.E. and Poso, H. (1983) S-Adenosylmethionine decarhoxylase (rat liver). Methods Enzymol., 94, 234-239. [I I] Erwin, B.G., Persson, L. and Pegg, A.E. (1984) Differential inhibition of histone and polyamine acetylases by multisubstrate analogues. Biochemistry, 23, 4250-4255. (121 Manni, A., Wright, C., Badger, B., Bartholomew, M., Herlyn, M., Mendelsohn, J., Masui, H. and Demers, L. (1990) Role of transforming growth factor-a-related peptides in the autocrine/paracrine control of experimental breast cancer growth in vifm by estradiol, prolactin, and progesterone. Breast Cancer Res. Treat., 15, 73-83. [I31 Fogel-Petrovic, M., Shappcll, N.W., Bergeron, R.J. and Porter, C.W. (1993) Polyamine and polyamine analog regulation of spermidine/spermine N’-acetyltransfemse in MALME-3M human melanoma cells. J. Biol. Chem. 268, 19118-19125. [14] Manni, A., Wechter, R., Grove, R., Wei, L., Mattel, J. and Demers, L. (1995) Polyamine profiles and growth properties of omithine decarboxylase overexpressing MCF-7 breast cancer cells in culture. Breast Cancer Res. Treat., 34, 4.5-53.
CancerLetters95 (1995)23-28
CANCER LETTERS
Isolation and characterization of human breast cancer cells overexpressing S-adenosylmethionine decarboxylase Andrea Man&*, Betty Badgera,Rhea Grovea,SusanKunselmanb,Laurence Demewc aDepartment of Medicine, Pennsylvania State University School of Medicine, 500 University Drive, P. 0. Box 850, Hershey, PA 17033-0850, USA bCenrerfor Biostatistics and Epidemiology, Pennsylvania State University School ofMedicine. 500 University Drive, P.O. Box 850, Hershey, PA 17033-0850, USA ‘Department qf Pathology, Pennsylvania State University School qf Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17033-0850, USA
Received10May 1995;accepted24May 1995
Abstract
We report the first successful isolation and initial characterization of S-adenosylmethioninedecarboxylase (SAMDC)overexpressingcells using a transfection approach.Stably transfectedMCF-7 breastcancercells overproducing SAMDC (-5fold) manifested reduced omithine decarboxylase while levels of N’-spermidinelspermine acetyltransferasewere variably increased.Analysis of cellular polyamine profile showed that spertnine was selectively increased(-80%), while spermidine and putrescine levels were reduced (-48% and -15% of control, respectively). Since SAMDC-overexpressing clones exhibited increasedclonogenicity in soft agar, our data suggestthat spermine may be selectively involved in conferring a more invasive phenotype to breastcancercells. Keywords: Human breast cancercells; S-Adenosylmethioninedecarboxylase;Polyamines; Soft agar clonogenicity; Proliferative activity
1. Introduction
Polyamines (putrescine, spermidine, and spermine) and their biosynthetic enzymes play an important role in carcinogenesis and neoplastic cell proliferation [I]. Data from our and other laboratories have * Correspondingauthor,Division of Endocrinology,Diabetes and Metabolism,PO Box 850, MS. HersheyMedical Center, Hershev.PA 17033.USA. Tel.: +l 717 5318395;Fax: +l 717 5315726.
demonstrated a critical role for polyamines in breast cancer growth [2]. Furthermore, in hormone-responsive mammary tumors, polyamines appear to be distal effecters of endocrine regulated cell proliferation
PI. Among the critical enzymes controlling the polyamine metabolic pathway, ODC has been the major target for investigation. The availability of several ODC over-producing cell lines resulting from either chronic DEMO exposure [3] or gene transfection [4] has been useful in analyzing the biochemical and
0304-3835/95/$09.500 1995ElsevierScienceIrelandLtd. All rightsreserved SSDI 0304-3835(95)03860-Y
24
A. Manni et al. I Cancer Letters 95 (I 9!?5) 23-28
biological properties of this enzyme. SAMDC, on the other hand, has been implicated to play a predominant role in tumor growth by promoting the formation of the more distal polyamines spermidine and spermine. As a result of this recognition, a recent impetus has been placed on the development of potent SAMDC inhibitors which have been shown to have marked anti-proliferative effects in numerous tumor models [5] including breast cancer [6]. However, in marked contrast to ODC over-producing cell lines, there is a paucity of SAMDC overexpressing cells which can be used to define the biological importance of this enzyme. To the best of our knowledge, there have been only two recent reports of SAMDC over-producing cells generated through selective pressure with an enzyme inhibitor [7,8]. To date, however, no stable overexpression of SAMDC by transfection has been achieved in any experimental system. In this paper, we report for the first time the successful isolation as well as initial characterization of stably transfected breast cancer cells overexpressing SAMDC. 2. Materials and methods 2. I. Cell lines and culture conditions Wild-type MCF-7 breast cancer cells (kindly provided by Dr. V.C. Jordan, University of Wisconsin, Madison, WI) and the transfected clones were propagated in 752 cm flasks in Richter’s improved minimal essential medium (IMEM) (Grand Island Biological, Grand Island, NY) containing phenol red and 5% FBS in a humidified atmosphere of 95% air, 5% CO, at 37°C. In selected experiments (see below), the culture conditions were stepped down to phenol red free IMEM containing dextran-coated charcoal stripped 5% FBS or to serum-free media. 2.2. Plasmid and transfection technique A 1.8 kb human SAMDC cDNA subcloned into an SV40-driven pCD expression vector [9] was kindly provided by Dr. A.E. Pegg (M.S. Hershey Medical Center, Hershey, PA). Transfection of MCF7 cells with pCD SAMDC and an RSV-neo cDNA (ratio 5:l) was carried out using a modified calcium phosphate precipitation technique as previously described by us [4]. Following G-418 selection, we isolated and expanded three control (pCD-3, -5, and
-6) and three SAMDC overexpressing clones (SAM1, -2, and -3). 2.3. Biochemical determinations pCD and pCDSAMDC-transfected clones were grown to near confluency in regular growth medium. For determination of enzymatic activities, the cells were washed three times with ice-cold PBS and then resuspended in buffer containing 50 mM Tris, 2.5 mM DTT, 0.1 mM EDTA (pH 7.5) and stored at -70°C until use. At the time of the assays, the cells were frozen and thawed twice. The cell lysates were centrifuged at 13 600 X g for 20 min. Activities of ODC, SAMDC and SSAT were determined in the cytosolic fraction according to standard techniques in routine use in our laboratory [4,10,11]. For measurement of polyamines, the cells were resuspended in 0.2 N perchloric acid and kept at 4°C overnight. The suspension was then centrifuged at 800 X g for 15 min and supernatant stored until the time of the assay. Polyamines were determined by HPLC with fluorometry as previously described [4]. 2.4. Riological studies Control and SAMDC overexpressing MCF-7 cells were compared with regard to anchorage dependent and independent growth. Plating in soft agar was done as previously published by us [ 121. Cells were plated in triplicate at a density of 1 X lo4 cells/ 35 mm dish. The number of colonies (aggregates > 50 cells) was scored after 20 days. Basal proliferative activity in liquid culture was assessedby plating cells in duplicate at a density of 6 X lo4 cells/35 mm dish in regular growth medium. Twenty-four hours later, the medium was replaced with either the same or phenol red free IMEM containing DCC-treated 5% FBS or serum-free IMEM supplemented with 5 mM glutamine, 0.2 mg% transferrin, 0.1 mg% fibronectin, 0.4 g% fraction V BSA, and 20 mM HEPES buffer. Duplicate dishes per experimental group were harvested on days 2, 4, and 6 and the number of cells was counted using a Colter Counter. 2.5. Statistical methods Analysis of variance (ANOVA) models were used to compare control and SAMDC transfected clones for enzymatic activities and polyamine levels (Table 1). Transformations of enzyme/polyamine levels
A. Munni et ~11.I Cuncer Letters 95 (I 995) 23-28
25
control). ODC activity was markedly suppressed to levels -20% of those detected in control cells. SSAT levels were found to be consistently higher in SAMDC overexpressing breast cancer cells. However, the magnitude of the increase was highly variable from experiment to experiment (see footnote to Table 1). Analysis of cellular polyamine profiles revealed a selective accumulation of spermine (-80% increase over control) while both putrescine and spermidine contents were decreased (-15% and -48% of control, respectively) (Table I). The increase, in spermine probably accounts both for the suppression of ODC and stimulation of SSAT activities. Spermidine and putrescine formed through the back conversion pathway controlled by SSAT are likely to be metabolically converted back to spermine by the increased SAMDC activity. Analysis of the biological properties of our clones revealed that SAMDC overexpressing MCF-7 cells formed more colonies in soft agar (Table 2). This growth advantage was manifested by all three clones and was highly significant in two experiments (experiments 2 and 3) while in experiment 1, only SAM-I exhibited increased clonogenicity. The influence of SAMDC overexpression on an-
were necessary in several cases to meet model assumptions of homogeneous variance and normality. All models included fixed effects for transfection type (vector only versus SAMDC) and experiment and the interaction between the two variables, where significant. For the soft agar experiments reported in Table 2, an ANOVA model was used, taking the square root of the colony number as the response to meet model assumptions. The model included fixed effects for transfection type, experiment and their interaction. Anchorage dependent growth was assessed by linear models which included harvest day, experiment and transfection type. A log transformation was performed on the cell counts to stabilize variance. Second order polynomials were used to model growth over time. All P values correspond to appropriate F-tests. All models were fit using the current version of the SAS statistical software package. 3. Results As can be seen in Table I, significant alterations in the polyamine metabolic pathway resulted from induction of SAMDC overexpression (-5-fold over
Table I Cellular levels of enzymatic activities and polyamines in control (pCD-3, 5, 6) and SAMDC-transfected cmcer cells” Clone
SAMDC (nmol/mg)
ODC (nmol/mg)
SSAT (pmol/mg)
pCD-3
0. I6 2 0.09
I .75 2 0.73
12.94 + 2.9
pCD-5
(10) 0.21 + 0.10
(3) 1.26kO.18
(3) 1.42 + I .6
(12)
(3) 2.59 (3) 0.41 (4) 0.42 (4) 0.33 (4)
(2)
pCD-6 SAM-1
0.17 + 0.07 (11) 0.85 ~0.18
(12) SAM-2 SAM-3
0.87 + 0.22 (11) 0.73 -co.18 (10)
r 1.31 rt 0.23 k 0.20 + 0.17
13.17 + (3) 22.21 * (4) 24.71 + (4) 24.55 t (4)
3.4 7.3 17.2 16.9
(SAM-l,
-2, -3) MCF-7 breast
Polyamines (nmol/mg) F’utrescine
Spermidine
Spermine
4.06 (4) 3.99 (4) 4.77 (4) 0.57 (4) II.62 i4) 0.77 ((4)
14.71 -t- 1.26 (4) 14.18 f 0.87 (4) 14.95 t 1.41 (4) 6.36 r 0.68 (4) 7.54 + 2.98 (4) 7.34 * I .04 (4)
I I .06 + 0.52 (4) 9.31 c 0.4s (4) IO.21 + 1.29 (4) 16.81 2 2.31 (4) 18.76 + 2.39 (4) 19.98 + 2.68 (4)
f 0.4.5 + 0.53 r0.14 * 0.30 + 0.48 + 0.3 1
a Data represent means k SD. Number of determinations is indicated in parentheses. The differences in levels of ODC and SAMDC activities and individual polyamines between control and SAMDC transfected clones were statistically significant (P < 0.0001). The increase in SSAT activity in SAMDC overexpressing cells was variable, being significant in one experiment (P= 0.002) but only marginal in another (P = 0.14).
26
A. Manni et al. I Cancer Letters 95 (1995) 23-28
Table 2 Anchorage independent growth of control and SAMDCoverexpressing MCF-7 breast cancer cellsa Clones
pCD-3 pCD-5 pCD-6 SAM-l SAM-2 SAM-3
Colony number Expt. 1
Expt. 2
Expt. 3
ND 278+ 18 203i7 404*10 270 k 35 192+28
118-+41 135+9 170 k 22 320 + 58 383 f 10 420 f 26
117+20 105 rt 16 155 + 18 241 + 14 201 k 11 259 f 27
12.8
12.6
c 8 1E e z s
A. /
.*-
12.4 -
12.2
-
2
3
a Data represent meansk SD of triplicate dishes/experimental condition. ND, not done. The differences in colony number between control and SAMDC-overexpressing cells were significant in Expts. 2 and 3 (P < 0.0001) but not in Expt. 1 (P = 0.15).
chorage-dependent growth was complex and depended upon the culture conditions. In the presence of regular growth medium, no major differences in proliferative activity were observed between control and SAMDC overexpressing cells (data not shown). When tested under steroid-depleted conditions, SAMDC overproducing cells grew at a faster rate than control (P = 0.04) (Fig. 1A). On the other hand, cell loss, which uniformly occurred under serum-free media conditions, was also significantly enhanced (P = 0.003) by SAMDC overexpression (Fig. 1B). 4. Discussion SAMDC is an enzyme of major importance in polyamine metabolism. It has been proposed that instead of ODC, SAMDC is the rate-limiting enzyme in the formation of spermidine and spermine [3], the polyamines most critically involved in cell proliferation. Hence, evaluation of the biochemical and biological function of this enzyme is of fundamental importance for improving our understanding of polyamine involvement in normal and neoplastic cell growth and for identifying new targets for anti-tumor therapy. The paucity of SAMDC overexpressing experimental systems has limited the investigation of the biochemistry and biology of this enzyme. Such experimental tools would be likely to be useful as shown by the well-proven value of numerous ODC overproducing cells for the study of ODC regulation and function [3,41.
4
6
5
Day
c 3 1c =I 8
10.0-
!! 9.5
-
l .
L 2
-.
l .
l .
3
Day 4
5
lo 6
Fig. 1. Anchorage dependentgrowth of control () (pCD-3, -5, -6) and SAMDC overexpressing(------) (SAM-l, -2, -3) MCF7 breast cancer cells cultured in either DCC-stripped serum contaming medium (A) or serum free medium (B) as specified in Section 2. The data represent the mean log (cell number) from 3 separateexperiments each done in duplicate, In every experiment, all 6 clones were tested.
We report here the first successful generation of SAMDC overexpressing cells using a transfection approach. This provided us with the opportunity to analyze the biochemical changes in the polyamine pathway produced by SAMDC overexpression. As might be anticipated on the basis of the well-known compensatory rise in ODC occurring in response to SAMDC inhibition [5], we observed a significant decrease in ODC activity in our SAMDC overproducing cells. Contrary to our results and surprisingly, ODC activity was found to be either slightly increased or unchanged in mouse FM3A [7] and CHO cells [8] overexpressing SAMDC following chronic
A. Manni et al. I Cancer Letters 95 (1995) 23-28
exposure to enzyme inhibitors. SSAT was found to be increased in our transfected clones, although the magnitude of this increase was variable from experiment to experiment. This finding is in agreement with the well known induction of this enzyme by polyamines [ 131.In our cells, we observed indeed a selective accumulation of spermine which we believe was responsible for the stimulation of SSAT activity. This enzyme, however, did not appear to be increased in SAMDC overexpressing CHO [8] cells and its activity was not reported in mouse FM3A cells [7]. Therefore there appear to be significant differences with regard to the changes in ODC and possibly SSAT activities induced by SAMDC overexpression between our MCF-7 cell clones and the FM3A and CHO cells. Whether these differences are due to the different approach employed to induce SAMDC overexpression (i.e. transfection versus selective pressure with an enzyme inhibitor) or reflect diversity in the intrinsic biologic properties of the cells remain to be determined. The selective increase in cellular spermine associated with a concomitant decrease in putrescine and spermidine contents is consistent with the distal activation of the polyamine pathway induced by SAMDC overexpression which leads to the accumulation of the final product spermine. A similar polyamine profile was also observed in SAMDC overproducing FM3A and CHO cells L',81. Our long-standing interest in polyamines focuses on their role in breast cancer biology. Since polyamines are critically involved in breast cancer cell proliferation and appear to be distal effecters of hormonally stimulated growth [2], we hypothesize that constitutive activation of the polyamine pathway may provide breast cancer cells with a growth advantage and possibly contribute to tumor progression. To test this hypothesis, we used a transfection approach to induce overexpression of key polyamine biosynthetic enzymes in breast cancer cells [4,14]. In previous experiments testing the role of ODC, we observed that ODC overexpressing breast cancer cells manifested a modest growth advantage in liquid culture and reduced sensitivity to estrogens[4,14]. The latter finding suggestsa role of polyamines in the transition of breast cancer to hormone independence. Clonogenicity in soft agar, however, was markedly reduced as a result of ODC overexpression [4]. In marked
21
contrast, as shown here, SAMDC overproducing breast cancer cells exhibited a significant growth advantagein soft agar. This difference probably reflects the different polyamine profiles induced by ODC versus SAMDC overexpression. While ODC overexpressing clones were characterized by selective accumulation of putrescine [4,14], SAMDC overproducing breastcancer cells manifested a selective increasein spermine. Since clonogenicity in soft agar correlates with tumor aggressiveness,these data suggestthe spermine may be selectively involved in conferring a more invasive phenotype to breast cancer cells. The influence of SAMDC overexpression and hence spermine accumulation on anchoragedependent growth was complex and depended upon the culture conditions. When tested in steroiddepletedmedia, SAMDC overexpressingclones grew faster than control cells. On the other hand, cell loss observed in all groups in serum-free media, also occurred at a faster rate in SAMDC transfectedMCF-7 cells. Additional studies will be required to define the mechanismssubservingthesecellular events. Acknowledgments This work is supportedby grant 5 PO1 CA4001 l10 from the National Cancer Institute. References ill Pegg, A.E. (1988) Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res.,48,159-114. PI Manni, A. (1994) The role of polyamines in the hormonal control of breast cancer cell proliferation. In: Mammary Tumorigenesis and Malignant Progression, pp. 209-225. Editors: R. Dickson and M. Lippman. Kluwer Academic, Nonvell, MA. [31 Pegg, A.E., Secrist III, J.A. and Madhubala, R. (1988) Properties of L1210 cells resistant to a-difluoromethylomithine. CancerRes., 48,267&2682. [41 Manni, A., Wechter, R., Wei, L., Heitjan, D. and Demers, L. (1995) Phenotypic features of breast cancer cells overexpressing omithine-decarboxylase. J. Cell. Physiol., 163, 129-136. PI Regenass,U., Mett, H., Stanek, J., Mueller, M., Kramer, D. and Porter, C.W. (1994) CGP 48664, a new Sadenosylmethionine decarboxylase inhibitor with broad spectrum antiproliferative and antitumor activity. Cancer Res.,54.3210-3217.
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A. Munni et al. I Cuncer Letters 95 (1995) 23-28
[6] Manni, A., Badger, B., Wechter, R. and Demers, L. (1995) Biochemical and growth modulatory effects of the new Sadenosylmethionine decarboxylase inhibitor CGP 48664 in breast cancer cells in culture. Submitted to the 86th Annual Meeting of the American Association for Cancer Research, Toronto, Canada. [7] Suzuki, T., Sadakata, Y., Kashiwagi, K., Hoshino, K., Kakinuma, Y., Shirahata, A. and Igarashi, K. (1993) Overproduction of S-adenosylmethionine decarboxylase in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A cells. Eur. J. Biochem., 215,247-253. [S] Kramer, D., Mett, H., Evans, A., Regenass, U., Diegelman, P. and Porter, C.W. (1995) Stable amplification of the Sadenosylmethionine decarboxylase gene in Chinese hamster ovary cells. J. Biol. Chem., 270,2124-2132. [9] Pajunen, A., Crozat, A., Janne, O.A., lhalainen, R., Laitinen, P.H., Stanley, B., Madhubala. R. and Pegg, A.E. (1988) and regulation of mammalian SStructure adenosylmethionine decarboxylase. J. Biol. Chem., 263, 17040-17049.
1101Pegg, A.E. and Poso, H. (1983) S-Adenosylmethionine decarhoxylase (rat liver). Methods Enzymol., 94, 234-239. [I I] Erwin, B.G., Persson, L. and Pegg, A.E. (1984) Differential inhibition of histone and polyamine acetylases by multisubstrate analogues. Biochemistry, 23, 4250-4255. (121 Manni, A., Wright, C., Badger, B., Bartholomew, M., Herlyn, M., Mendelsohn, J., Masui, H. and Demers, L. (1990) Role of transforming growth factor-a-related peptides in the autocrine/paracrine control of experimental breast cancer growth in vifm by estradiol, prolactin, and progesterone. Breast Cancer Res. Treat., 15, 73-83. [I31 Fogel-Petrovic, M., Shappcll, N.W., Bergeron, R.J. and Porter, C.W. (1993) Polyamine and polyamine analog regulation of spermidine/spermine N’-acetyltransfemse in MALME-3M human melanoma cells. J. Biol. Chem. 268, 19118-19125. [14] Manni, A., Wechter, R., Grove, R., Wei, L., Mattel, J. and Demers, L. (1995) Polyamine profiles and growth properties of omithine decarboxylase overexpressing MCF-7 breast cancer cells in culture. Breast Cancer Res. Treat., 34, 4.5-53.