ARTICLE |
Correspondence to: David J. Uhlinger, Johnson & Johnson Pharmaceutical Research & Development, Raritan, NJ 08869. E-mail: duhlinger@prdus.jnj.com
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Summary |
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Metastatic processes, including cell invasion, extracellular matrix degradation, and tissue remodeling, require cellular reorganization and proliferation. The cell signaling molecules required and the proteins involved in cell restructuring have not been completely elucidated. We have been studying the role of sphingolipids in normal cell activity and in several pathophysiological states. In this study we used immunohistochemistry to observe the presence of the two known subunits of serine palmitoyltransferase (SPT) in proliferating cells, in an in vitro model of wound repair, and in human malignant tissue. We report increased expression of the two subunits, SPT1 and SPT2, in the proliferating cells in these models. We also demonstrate a change in subcellular localization of the SPT subunits from predominantly cytosolic in quiescent cells to nuclear in proliferating cells. In addition, we observed SPT1 and SPT2 immunoreactivity in reactive stromal fibroblasts surrounding the carcinoma cells of some of the tumors. This enhanced SPT expression was absent in the stromal fibroblasts surrounding normal epithelial cells. Our results suggest a potential role for overexpression of SPT in the processes of cell metastasis.
(J Histochem Cytochem 51:715726, 2003)
Key Words: serine palmitoyltransferase, metastatic carcinomas, macrophages, immunohistochemistry, reactive fibroblasts, tumor microenvironment
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Introduction |
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COMPLEX SPHINGOLIPIDS are produced via an intricate metabolic pathway from which many bioactive intermediates are generated. Regulation of the enzymes involved in sphingolipid metabolism via external stimuli including UV light (
Serine palmitoyltransferase (SPT) is the initial and rate-limiting enzyme in the de novo sphingolipid biosynthesis cascade. At least two subunits are involved in the enzymatic activity associated with SPT, SPT1 and SPT2 (
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Changes in lipid metabolic pathways are among the first events in the de-differentiation of normal cells (
The role of nuclear lipid metabolism in signal transduction cascades has recently become apparent. Diacylglycerol kinase, an enzyme involved in phospholipid metabolism, has been shown to localize to the nucleus and is involved in nuclear signal transduction (
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Materials and Methods |
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Antibody Characterization and Western Blotting
Rabbit polyclonal antibodies to the SPT subunits SPT1 and SPT2 have been characterized and evaluated previously (
Immunohistochemistry (IHC)
Table 1 lists the titer and source of the positive and negative control antibodies and the experimental antibodies used in these studies. Commercial human normal and tumor checkerboard tissue slides (DAKO, Carpenteria, CA; Biomeda, Foster City, CA) were deparaffinized, hydrated, and processed for routine IHC, which has previously been described (
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Cell Culture
Human neonatal dermal fibroblasts and their culture media were obtained from Clonetics/BioWhittaker (Walkersville, MD). Cell suspensions (5 x 104/ml) were seeded in four-well chamber slides (NUNC; Naperville, IL) for immunocytochemistry. To mimic the in vivo activation of differentiated quiescent fibroblasts in vitro, a scrape-wounding model was used as described previously (
Immunocytochemistry (ICC)
Four-chambered cultured slides were fixed with 10% neutral buffered saline for 10 min at RT, rinsed in PBS, and then assayed for ICC as previously described (
Double Immunohistochemistry (IHC:IHC)
To determine if the SPT subunits co-localized in proliferating cells, we used double immunohistochemical methods (IHC:IHC) to simultaneously detect SPT1 or SPT2 expression with detection of a proliferation marker, proliferating cell nuclear antigen (PCNA), using protocols described previously (
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Results |
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SPT catalyzes the initial and rate-limiting step in de novo sphingolipid synthesis, as shown in Fig 1. This biosynthetic pathway generates ceramide, which serves as a second messenger signaling molecule and as the precursor molecule for complex sphingolipids.
To characterize the expression and localization of SPT1 and SPT2 in cells and tissues by ICC and IHC, antibodies specific to SPT1 and SPT2 (
Subconfluent human dermal fibroblasts were immunolabeled by ICC to observe the expression and localization of SPT1 and SPT2 in the cells. Fig 2 shows the results of the negative control labeling (Fig 2A), PCNA labeling (Fig 2B), which appears as nucleus-associated labeling in the proliferating cells, SPT1 (Fig 2C) and SPT2 (Fig 2D) labeling. Interestingly, although some diffuse staining was observed throughout the fibroblasts, the majority of the labeling by the anti-SPT antibodies appeared to be associated with the nucleus in these proliferating fibroblasts. There were, however, nuclei that were not stained by PCNA, SPT1, or SPT2 antibodies.
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An in vitro model was used to mimic the in vivo activation by tissue wounding of differentiated quiescent fibroblasts to further characterize the expression of SPT in proliferating cells. Quiescent fibroblast cultures were compared to confluent cultures subjected to mechanical scraping and allowed to recover for 5 days (wound conditions). Fig 3 shows no labeling by ICC in the 9-day quiescent cultures (Fig 3A, Fig 3D, Fig 3G, and Fig 3J) or the 14-day quiescent cultures (Fig 3B, Fig 3E, Fig 3H, and Fig 3K) immunolabeled with the negative control (Fig 3A and Fig 3B) and PCNA (Fig 3D and Fig 3F) antibodies. Light diffuse labeling was observed with the SPT1 (Fig 3G and Fig 3H) and SPT2 (Fig 3J and Fig 3K) antibodies. In contrast, the 14-day wounded fibroblasts (Fig 3C, Fig 3F, Fig 3I, and Fig 3L) showed strong immunolabeling for PCNA (Fig 3F), SPT1 (Fig 3I), and SPT2 (Fig 3L), and no staining in the negative control (Fig 3C). The most dramatic observation was the intense nuclear localization of SPT1 and SPT2 in the 14-day wounded fibroblasts (Fig 3I and Fig 3L) in comparison to the 9 (Fig 3G and Fig 3J)- and 14 (Fig 3H and Fig 3K)-day quiescent cells.
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To demonstrate an increase in SPT1 and SPT2 proteins in the nuclei of proliferating neonatal fibroblasts, Western blots were performed with equal concentrations of nuclear extracts from quiescent and proliferating dermal fibroblasts, respectively (Fig 4) SPT1 and SPT2 were more abundant in the nuclear protein extract from proliferating cells than that from quiescent cells. The increase in SPT2 in proliferating cells compared with quiescent cells was more dramatic than that observed for SPT1. In competition assays performed simultaneously, the presence of antigenic peptides used for generation of the SPT1 and SPT2 antibodies completely inhibited SPT1 (Fig 4, Lanes 4 and 5) and SPT2 (Fig 4, Lanes 9 and 10) detection.
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To investigate the lack of staining seen in some nuclei (Fig 2 and Fig 3), double staining (IF:IF) (Fig 5) was used to show coincidence of PCNA (Fig 5B and Fig 5D) and increased SPT1 (Fig 5A) and SPT2 (Fig 5C) labeling. Arrows indicate cells in which PCNA was detected, and the SPT labeling appeared to be strong and associated with the nucleus. Arrowheads indicate cells lacking PCNA-label and with diffuse SPT labeling that did not show specific association with the nucleus. From the expression patterns observed in this study and the previously described in vitro wounding model, it appears that SPT expression is increased and nucleus-associated in proliferating cells. In addition to the model of wounding described above, we have recently reported the increased expression of SPT1 and SPT2 in de-differentiated fibroblasts and proliferating vascular smooth muscle cells in balloon-injured rat carotid artery (
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Parallels between wound repair responses and tumor formation have recently become even more apparent (
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Table 2 shows the immunolocalization of SPT1 and SPT2 in a variety of human malignancies. Positive labeling was defined by the presentation of brown staining and was scored according to the number of labeled cells for SPT1 and SPT2 in a x100 viewing field in 18 different human tumors with an average of five fields of each tissue: negative (N) = no labeled cells; weak (W) = 110 labeled cells; moderate (M) = 1120 labeled cells; strong (S) = >20 labeled cells. Five carcinomas (colon, ovarian, pancreas, thyroid, undifferentiated) and two sarcomas demonstrated strong overexpression of both SPT subunits. Other tumors (Table 2) expressed little (W) or no (N) detectable levels of SPT1 or SPT2.
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Human malignant colon carcinoma tissues were processed for IHC using antibodies to the preimmune serum (Fig 7A), SPT1 (Fig 7B), and SPT2 (Fig 7C). Fig 7 also shows strong intracellular labeling of SPT1 (small arrowheads) and SPT2 (small arrowheads) in the malignant cells. In addition to the labeling observed in the malignant cells, immunostaining was observed in the stromal fibroblasts adjacent to the tumor (large arrowheads). Human malignant undifferentiated carcinoma tissues (Fig 7D7F) were processed for IHC using antibodies to the preimmune serum (Fig 7D), SPT1 (Fig 7E), and SPT2 (Fig 7F). There appeared to be a stronger SPT1 signal (arrowheads, Fig 7E) in the undifferentiated human carcinoma tissue relative to the weak SPT2 signal (Fig 7F). These slides were processed simultaneously in a multi-tissue format. Therefore, comparisons of the intensity of the signal among the tissue samples may be relevant. Because the role played by the two SPT subunits in the enzymatic activity, whether catalytic or regulatory, is still unclear, it is not evident whether this enhanced SPT1 expression, or even the ratio of SPT1 to SPT2 expression, is critical to a cell response.
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Fig 7G7I show the observations made in human malignant thyroid carcinoma tissues processed using the preimmune serum (Fig 7G), SPT1 (Fig 7H) and SPT2 (Fig 7I) antibodies. There appeared to be SPT-specific staining in the majority of the cells of this tumor. The intensity of the SPT1 and SPT2 signal (arrowheads) is variable from cell to cell, with some of the malignant cells expressing very high levels of the SPT subunits. Further studies are needed to determine if the various expression levels of these subunits indicate different requirements for the enzyme in tumor cells.
In addition to studying various carcinoma tissue samples, we also examined human malignant sarcoma tissues (Fig 7J7L). We observed abundant expression of SPT1 and SPT2 (arrowheads) in this malignancy as well as in the carcinomas described above. These results indicate that the overexpression of SPT in cancer cells is not limited to those derived mostly from epithelial cells, such as carcinomas. Cancers such as sarcomas, which are derived from mesenchymal cells, also appear to have induced expression of the SPT subunits.
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Discussion |
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We have developed antibodies to human SPT1 and SPT2, which have enabled us to investigate the expression of these enzyme subunits in normal human tissue (
Because of the emerging paradigm that some of the key molecules involved in the cell wound repair response are also involved in tumor growth and metastasis, we performed a series of IHC studies on tumor cell lines and human tumor tissues to study SPT1 and SPT2 expression. The SPT subunits are abundantly expressed in several well-established human tumor cell lines, including a lymphoma, an adenocarcinoma, and a neuroblastoma cell line. We provide morphological evidence for increased expression of SPT1 and SPT2 in malignant carcinoma cells and in the cell types forming the tumor microenvironment (TME), such as the reactive stromal fibroblasts and local macrophages. SPT1 and SPT2 were not detected in stromal fibroblasts in similar normal tissues (
Our lab and others have observed enhanced expression of SPT in activated leukocytes involved in an inflammatory response (
Sphingosine-1 phosphate (S-1-P), a sphingolipid that can be generated via the de novo biosynthetic pathway (Fig 1), has clearly been shown to act as a mitogen through intra- and intercellular signaling (
Other sphingolipids in addition to S-1-P that can be generated via the de novo synthesis pathway have been implicated in proliferative and metastatic processes. Changes in expression of various glycosphingolipids on the cell surface have been correlated with acquiring and maintaining cancer phenotypes, tumor progression, and metastasis (
Elevated levels of glycosphingolipids in cell membranes may bring about changes in cellcell interactions and could modulate receptor complex formation and signal transduction through membrane receptors. Studies are under way to determine if glycosphingolipid content is being modulated through regulation of de novo sphingolipid synthesis in malignant cells and in the cells that make up the TME. A number of studies have looked at sphingolipid metabolites, particularly ceramide, and their relationship to the effectiveness of various cancer therapeutics (
Additional studies will be needed to determine if SPT levels and cell location correlate with specific tumor types or if the relative amounts of SPT1 and SPT2 in the tumor cells and in the stromal fibroblasts are clinically relevant. It is possible that the amounts of SPT1 and SPT2 in the TME cells may be a valid predictor of metastatic activity, thereby imparting diagnostic and prognostic value. More importantly, these data suggest that serine palmitoyltransferase inhibitors may represent a novel class of compounds with therapeutic utility against certain neoplasias.
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Acknowledgments |
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We express our gratitude for the excellent histological and immunohistochemical expertise of Patti A. Reiser, BS, MT, HT (ASCP), Norah A. Gumula, HT (ASCP), Brenda M. Hertzog, BS, MT (ASCP), Debbie A. Polkavitch, BS, MT (ASCP), Barbara Branchide, and Danielle Schmidheiser, BS, of the MorphoMetrics department.
Received for publication April 24, 2002; accepted January 22, 2003.
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