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Malignant Transformation Alters Membrane Choline Phospholipid Metabolism of Human Mammary Epithelial Cells¹

The Johns Hopkins University School of Medicine, Division of Magnetic Resonance Research–Oncology Section, Department of Radiology, Baltimore, Maryland 21205

	ABSTRACT

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Transduction of mitogenic signals in cells can be mediated by molecules derived from the synthesis and breakdown of the major membrane phospholipid, phosphotidylcholine. Studies were performed on human mammary epithelial cells in culture to understand the impact of malignant transformation and progression on membrane phospholipid metabolism. In the model system used here, phosphocholine levels and total choline-containing phospholipid metabolite levels increased with progression from normal to immortalized to oncogene-transformed to tumor-derived cells. These changes occurred independently of cell doubling time. A "glycerophosphocholine to phosphocholine switch" was apparent with immortalization. This alteration in phenotype of increased phosphocholine relative to glycerophosphocholine was observed in oncogene-transformed and for all human breast tumor cell lines analyzed. The results demonstrate that progression of human mammary epithelial cells from normal to malignant phenotype is associated with altered membrane choline phospholipid metabolism.

	INTRODUCTION

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PtC³ is the most abundant phospholipid in biological membranes and together with other phospholipids, such as phosphatidylethanolamine and neutral lipids, form the characteristic bilayer structure of cells and regulate membrane integrity (1 , 2) . MCPM (Fig. 1) , i.e., biosynthesis and hydrolysis of PtC, are essential processes for mitogenic signal transduction events in cells (3, 4, 5, 6) . There is now increasing evidence to suggest that products of MCPM such as PCho, diacylglycerol, and arachidonic acid metabolites may function as second messengers essential for the mitogenic activity of growth factors particularly in the activation of the ras-raf-1-MAPK cascade and protein kinase C pathway (3, 4, 5, 6) . Together with inositol phospholipid metabolism, MCPM can also provide a sustained activation of mitogenic signal transduction via a positive feedback interaction (4 , 7) .

View larger version (19K): [in this window] [in a new window]   Fig. 1. Biosynthesis and hydrolysis of PtC. Phosphorylation of choline to PCho by choline kinase (CK) is the first step in the biosynthesis of PtC. PCho is then converted to PtC via intermediates involving the rate-limiting enzyme CTP: phosphocholine cytidyltransferase (CT) and phosphocholine transferase (PCT). Hydrolysis of PtC is effected by three major PtC-specific enzymes, phospholipase C (PLC), phospholipase D (PLD), and phospholipase A1 and A2 (PLA1 and PLA2). FFA, free fatty acid.

NMR has been used to study choline phospholipid metabolism in cells or excised tissues, as well as noninvasively in vivo (10, 11, 12, 13) . Depending on the experimental conditions, ¹H NMR methods can detect either individual choline phospholipid metabolites or a peak corresponding to total choline-containing metabolites. Using ¹H NMR, invasive cancer could be distinguished from benign breast lesions by the high total choline phospholipid metabolite levels in the former (10) . In another study, increased choline phospholipid metabolite levels characterized two cancer cell lines (MCF-7 and T47D) compared with that of a normal HMEC line; there were no distinct differences in high energy phosphates and the rates of glucose consumption and aerobic glycolysis (14) . These studies support the existence of differences in phosphatidylcholine metabolism between normal epithelial cells and cancer cells in vitro and between benign and malignant cells in vivo. The possibility of differential regulation of MCPM in normal versus tumor cells suggests a diagnostic role for enhanced MCPM and has implications for therapeutic intervention.

Despite the indication of an altered MCPM in breast cancer cells, no attempt has been made to systematically relate the multistep process of carcinogenesis to altered MCPM in mammary epithelial cells. To address this issue, we have assessed PCho, GPC, and choline levels in a number of epithelial cell lines derived from reduction mammoplasty (normal) tissues and neoplastic lesions and also investigated the effects of immortalization and oncogene transformation on MCPM. Such a model has been used previously by other workers to evaluate the stepwise progression in mammary epithelium from normal to malignant phenotype (15, 16, 17, 18, 19, 20) . Our data suggest that phenotypic changes in MCPM probably commence early in carcinogenesis and may, as with most other neoplastic phenotypes, be regulated by an interplay of cellular immortalization and oncogene transformation.

	MATERIALS AND METHODS

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Cell Lines.
HMECs used in this study include finite life span HMEC strains 184 and 48, derived from reduction mammoplasty tissues; nontumorigenic immortal cell lines 184A1 and 184B5, derived from benzo(a)pyrene-treated 184 cells; and the 184B5-erbB2 cell line, derived from 184B5 by transfection with the erbB2 oncogene. All of the above cell lines were obtained from Dr. Martha Stampfer (Lawrence Berkeley National Laboratory, Berkeley, CA) and cultured in MCDB 170 media supplemented as described previously (21 , 22) . MCF-12A, a spontaneously immortalized cell line established from MCF-12M mortal cells (23) , was obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM-Ham’s F12 medium supplemented as described previously (23) . All of the human breast cancer cell lines were derived from pleural effusions in patients with breast cancer and were obtained from American Type Culture Collection. The tumor-derived cell lines were all cultured in DMEM-Ham’s F12 medium supplemented with 10% fetal bovine serum.

Growth Rate and Cell Size.
The growth rate of the cell lines used in this study were determined using the MTT assay (24) . Briefly, cells (5 x 10³) were plated in 24-well plates in 1 ml of media and incubated under normal culture conditions for up to 6 days. To estimate cell number, the cells were incubated with MTT (Sigma Chemical Co., St. Louis, MO) for 4 h. MTT was then removed, and the resulting formazan crystals were dissolved in 1 ml of DMSO and 125 µl of glycine buffer (pH 10.5; Ref. 24 ). The UV absorbance of the formazan solution was recorded at 553 nm (max). Four replicates were used to calculate the cell doubling time for each cell line. Because the cells had different morphologies and diameters, the cell size was determined for each cell line by trypsinizing the cells and counting the diameter of 20 random cells using an optical microscope.

Extraction.
To determine the choline phospholipid metabolite content, cells growing in culture were fed with fresh media 24 h before extraction and used at 70–80% confluency. Cells (10⁷ to 10⁸) were trypsinized, washed twice with normal saline, and homogenized with ice-cold 8% perchloric acid (5 ml). The homogenates were centrifuged (15000 rpm for 15 min at 4°C), and the supernatants were neutralized with 3 M K₂CO₃/1 M KOH buffer. The samples were again clarified by centrifugation, treated with 50 mg Chelex (Sigma) to remove divalent ions, lyophilized, and resuspended in 0.5 ml of D₂O for NMR analysis. Trimethylsilyl propionate (5 µl) was used as an internal standard. ¹H NMR spectra of the extracts were acquired on a 11.7T Bruker NMR spectrometer with a 5-mm probe. Fully relaxed spectra (without saturation effects) were obtained using the following acquisition parameters: 30° flip angle, 6000 Hz sweep width, 4.7 s repetition time, 32 K block size, and 512 scans. The data were analyzed using an in-house software, Soft Fourier Transform (P. Barker, The Johns Hopkins University). PCho, GPC, and total choline-containing (PCho + GPC + choline) metabolite levels were determined and normalized to cell size. Between three and five independent extracts were analyzed per cell line.

The reason for normalizing metabolite levels to cell size was due to differences in cell size between the cell lines used in this study. This necessitated normalization to either cell size or protein concentration. The former requires fewer cells and is therefore suited to experiments with mortal cells, which senesce after 5 to 18 passages. To determine concentrations, peak amplitudes for choline PCho, GPC, and total choline-containing metabolites (PCho + GPC + choline) were compared with that of the internal standard TSP according to the equation:

where [metabolite] is concentration of the metabolite expressed as fmol/µm³, [TSP] is the molar concentration of TSP used, and cell volume (µm³) was calculated from the radius of the cell according to the equation, volume = 4/3 x ³. For this equation to be valid, it is necessary that spectra are fully relaxed, as was the case here, or to correct for saturation.

Statistical Analysis.
Statistical analysis of the data were performed using StatView II version 1.04, 1991 (Abacus Concepts, Inc., Berkeley, CA). The statistical significance of differences in metabolite levels between cell lines was determined using the Mann-Whitney U test. Ps of 0.05 were considered to be significant.

	RESULTS

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Identification of Phospholipid Metabolites by ¹H NMR.
¹H NMR of perchloric acid extracts demonstrated the presence of three water-soluble, choline-containing [-N(CH3)₃] metabolites, i.e., choline, PCho, and GPC (Fig. 2) . These metabolites resonate at 3.2 ppm downfield of the internal standard and chemical shift reference TSP. Peak assignments were performed with authentic compounds. Ten epithelial cell lines of mammary origin were characterized by this method; the phenotype and cell size of these cell lines are indicated in Table 1 .

View larger version (12K): [in this window] [in a new window]   Fig. 2. Typical 1H NMR spectra obtained from perchloric acid extracts of MCF-12A (a) and MDA-MB-231 (b) breast cancer cells grown in culture. The spectra, expanded to show the choline-containing metabolite region, represent qualitative differences between the two cell lines, i.e., not normalized to display comparable signal-to-noise levels. Spectral assignments include GPC (3.234 ppm), PCho (3.225 ppm), and free choline(Cho; 3.207 ppm).

View larger version (17K): [in this window] [in a new window]   Fig. 3. PCho:GPC ratios in a panel of cell lines representing various stages of breast carcinogenesis. There was a statistically significant difference in PCho:GPC ratio (P < 0.05) between finite life span versus tumor-derived cells, 184 strain versus 184A1 cell line, and 184B5 versus 184B5-erbB2 cell lines. The P for 184 strain versus 184B5 cell line was 0.1. Bars, SE.

View larger version (14K): [in this window] [in a new window]   Fig. 4. PCho levels (a), GPC levels (b), and total choline-containing metabolite (PCho + GPC + choline) levels (c) in a panel of cell lines representing various stages of breast carcinogenesis. There was a statistically significant difference in total choline-containing metabolite levels and PCho levels (P < 0.05) for finite life span cells versus tumor-derived cells; 184 strain versus 184A1; 184B5 versus 184B5-erbB2; MDA-MB-435 versus MDA-MB-231, MCF7, and SKBR3; MDA-MB-231 versus SKBR3; and MCF7 versus SKBR3. There was a statistically significant difference in GPC levels for finite life span cells, 184A1, 184B5 and 185B5-erbB2, and SKBR3 versus MDA-MB-435, MDA-MB-231, MCF7, and MCF-12A cells. Bars, SE.

View larger version (18K): [in this window] [in a new window]   Fig. 5. The relationship between doubling time and PCho levels (a), GPC levels (b), total choline-containing metabolite levels (PCho + GPC + choline; c), and PCho:GPC ratio (d). Doubling times were measured by the MTT assay (see "Materials and Methods") and increased in the order MCF-12A > 48 (mortal) > MDA-MB-435 > 184B5-erbB2 > 184B5 > MDA-MB-231 > 184 (mortal) > 184A1 > MCF-7 > SKBR3; , MDA-MB-435; , MDA-MB-231; •, MCF-7; , SKBR3; , MCF-12A; , 184 (mortal); , 48 (mortal); , 184A1; , 184B5; , 184B5-erbB2. Bars, SE.

	DISCUSSION

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We have investigated the association between malignant carcinogenic processes and MCPM by monitoring the three choline phospholipid metabolites (choline, PCho, and GPC) in 10 cell lines, which represent different stages of malignant progression. Our findings suggest that normal human mammary epithelium has low steady-state levels of total choline-containing metabolites. In addition to their low total choline-containing metabolite levels, we also demonstrated that GPC was the major metabolite in the normal HMECs. A GPC to PCho switch appeared to be an early phenotypic change during carcinogenesis, as observed in benzo(a)pyrene-immortalized cells and where instead of GPC, PCho became the major choline phospholipid metabolites. However, despite this "switch," total choline-containing metabolite levels remained low in these immortalized cells. Transformation of 184B5 immortal cells by forced overexpression of the erbB2 oncogene, however, resulted in a dramatic increase in both PCho:GPC ratio and total choline levels compared with the benzo(a)pyrene-immortalized cells. However, total choline-containing metabolites and PCho levels were still less than those of tumor-derived cells. erbB2 is an important (proto)oncogene that is amplified in 20–30% of breast cancer cases and is associated with poor prognosis; amplification of this oncogene is thought to occur late in tumor progression (27, 28, 29, 30) . Transformation of 184B5 by this gene results in the ability of these cells to form colonies in semisolid medium and to form small, low frequency tumors with high latency in vivo (16) . Our data with erbB2 demonstrate a new and heretofore unknown metabolic role for erbB2 and support the possibility that growth factor-mediated activation of the tyrosine kinase cascade (involving receptor-grb 2-sos-ras-raf-1-MEK-MAPK) can lead to an increase in PCho levels (6, 7, 8, 9) . In general, the levels and expression of receptors and proteins involved in the growth factor receptor-tyrosine kinase pathway tend to increase with malignancy. For instance, levels of epidermal growth factor receptor are low in the 184 strain, moderately high in 184A1, 184B5, and 184B5-erbB2 cells, and very high in MDA-MB-231 cells (19 , 31) . In addition, Daly et al. (32) reported up-regulation of grb2 mRNA/protein and the ras signaling pathway in MCF-7 and MDA-MB-231 cells compared with normal HMECs.

All of the breast tumor cell lines showed the GPC to PCho switch. In addition to this switch, all breast tumor cells showed significantly higher total choline-containing metabolite levels (P < 0.05). The increased total choline-containing metabolite levels were mainly due to an increase in PCho levels and, to a lesser and variable extent, an increase in GPC levels. There was a gradual increase in both total choline-containing metabolite levels and PCho levels as the cells acquired malignant phenotype (normal < immortal < oncogene-transformed < tumor-derived), with the highly invasive metastatic cell lines showing the highest levels. The high total choline content in the tumorigenic cells may be related to the multiple genetic changes that are associated with the multistep process of carcinogenesis (28) and may explain the progressive ability of these cells to gain anchorage-independent growth, form primary tumors in immune compromised mice, and finally to metastasize. Our studies confirm the work of Ting et al. (14) , who showed for a limited number of cell lines that levels of choline-containing metabolites were low in a normal mammary epithelial strain and high in two tumor-derived cell lines. Our results also support recent clinical observations that the total choline peak is higher for malignant lesions than for benign ones (10) .

It has been postulated that the rapid growth and proliferation of cancer cells and increased membrane/fatty acid requirements may be responsible for the high choline phospholipid metabolite levels in cancer versus normal tissues (12 , 25 , 26) ; the same argument could be made for benign lesions versus invasive cancers. However, the data presented here and that of Ting et al. (14) show that choline-containing metabolite levels remain low in normal HMECs in culture when the cells are proliferating at approximately similar rates as tumor-derived cells and suggest that although proliferation-related changes may occur (26) , the rate of proliferation per se cannot completely account for the increased choline phospholipid metabolism. In this study, we have demonstrated that an alteration in MCPM is linked to malignant transformation and progression of mammary epithelium. Presently, the exact mechanisms underlying the altered metabolism are unknown. Possible mechanisms include activation of enzymes involved in MCPM, such as via enhanced receptor tyrosine kinase cascade (9) , or differential induction of choline kinase isozymes, as reported previously for carcinogen-treated rat liver (33) . Other possible mechanisms that need to be investigated include amplification of choline kinase, phospholipase C, phospholipase D, and phospholipase A genes during carcinogenesis.

To conclude, the major finding to emerge from the present study is that choline phospholipid metabolite levels progressively increase in cultured HMECs as cells become more malignant. We therefore propose that carcinogenesis in human breast epithelial cells results in progressive alteration of membrane choline phospholipid metabolism. This work is relevant to diagnosis of breast cancer and also provides a rationale for selective pharmacological intervention.

	ACKNOWLEDGMENTS

 
We are very grateful to Dr. Martha Stampfer of Berkeley National Laboratory (Berkeley, CA) for kindly donating the HMECs used in this study and for very useful suggestions. We gratefully acknowledge the expert technical assistance of Dr. V. P. Chacko in performing the NMR spectroscopy experiments, and we thank N. Mori for assistance with the cell doubling time measurements.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by USAMRMC Grant DAMD-17-96-16131.

² To whom requests for reprints should be addressed, at The Johns Hopkins University School of Medicine, Division of Magnetic Resonance Research–Oncology Section, Department of Radiology, 208C Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205.

³ The abbreviations used are: PtC, phosphatidylcholine; MCPM, membrane choline phospholipid metabolism; PCho, phosphocholine; GPC, glycerophosphocholine; NMR, nuclear magnetic resonance; HMEC, human mammary epithelial cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TSP, trimethylsilylpropionate.

⁴ Z. M. Bhujwalla and E. O. Aboagye, unpublished data.

Received 6/18/98. Accepted 10/28/98.

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