(CANCER RESEARCH 50. 6068-6074. September 15. 1990] A Comparison of Synaptophysin, Chromogranin, and L-Dopa Decarboxylase as Markers for Neuroendocrine Differentiation in Lung Cancer Cell Lines1
Sandra M. Jensen, Adi F. Gazdar, Frank Cuttitta, Edward K. Russell, and R. Ilona Linnoila2
National Cancer Institute-Navy Medical Oncology Branch, Clinical Oncology Program, Division of Cancer Therapy, National Cancer Institute Navy Hospital, Belhesda. Mary-land 20814
ABSTRACT NE tumors of the lung include bronchial carcinoid, SCLC-C, and SCLC-V cell types. It is important to distinguish SCLC Synaptophysin is a M, 38,000 integral membrane glycoprotein ex from other lung cancers because of the better therapeutic re pressed by a variety of normal and neoplastic neuroendocrine cells. \Ve studied Synaptophysin as an immunocytochemical marker for neuroen sponse to cytotoxic therapy in patients (3). Among SCLC, the docrine differentiation in lung cancer and compared it to the immunocy variant subtype SCLC-V is associated with worse prognosis tochemical expression of chromogranin A, a marker for dense core than SCLC-C. Although considered NE tumors, SCLC-V cell (endocrine) granules, and the biochemical activity of i.-dopa decarboxyl- types typically lack dense core granules and may, therefore, be ase (DDC), the key amine-handling enzyme. Of the 250 cell lines difficult to diagnose (4-6). available to us, we selected examples representative of the following cell ExPuSC is morphologically similar to SCLC and has been types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = described in a large variety of sites, including esophagus, stom 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell ach, pancreas, larynx, hypopharynx, salivary glands, nasal cav lung cancers (n = 18) whose neuroendocrine status had been previously ity and paranasal sinuses, thymus, small and large bowel, uter determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of Synaptophysin than ine cervix, endometrium, breast, prostate, urinary bladder, and chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic skin. A significant number of, but not all, ExPuSC tumors have SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (ft) been shown to contain neurosecretory granules and produce 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines ectopie hormones (which can be demonstrated using immuno- expressed Synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non- histochemical techniques) (7-9). We included four ExPuSC cell small cell lung cancer cell lines previously shown to express multiple lines in our study because of their similarities to NE tumors of neuroendocrine markers were positive for Synaptophysin, chromogranin the lung, namely SCLC. A, and DDC activity; (proteins ranging cytochemical techniques are employed. This subset of NSCLCs from U, 43,000 to 45,000 in five representative cell lines. The concord that express multiple NE markers has been named NSCLC- ance of expression of all three neuroendocrine markers was statistically NE(10, 11). significant when values for all cell lines were totalled. Synaptophysin was Specific NE markers in lung cancers consist of a large number a more commonly expressed marker for variant SCLC cell lines, which of defined hormones and neuropeptides, both eutopic (i.e., rarely showed DDC activity. We conclude that Synaptophysin may be a gastrin-releasing peptide, calcitonin, and serotonin) and ectopie more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines (adrenocorticotropic hormone, vasopressin, and neurotensin). which express only part of the neuroendocrine program. However, these NE products are seldom very useful for diag nosis, since their expression in tumors is quite variable and may be subtype specific (10). INTRODUCTION Among the currently available general NE markers are NSE, NE' tumors are a diverse group of neoplasms that usually Leu-7, CgA, and DDC (2, 10). Immunocytochemical and en express multiple markers for NE differentiation. They are char zymatic techniques have demonstrated these general NE mark acterized by the presence of dense core granules, high concen ers to be useful investigative and diagnostic tools, but not trations of the key amine-handling enzyme L-DOPA decarbox- without some limitations. NSE, the 7-subunit of the glycolytic ylase (aromatic-L-amino acid decarboxylase, EC 4.1.1.28), and enzyme enolase, was originally thought to be expressed exclu the production of various hormones and neuropeptides (1, 2). sively in neurons and NE cells (12). It is now known that NSE is expressed in a variety of non-NE cells and tumors, which has Received 1/20/89; accepted 6/18/90. led many investigators to question its specificity (13-15). Leu- 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 7, a differentiation antigen shown to be selectively expressed accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1The opinions and assertions contained herein are the private views of the on human natural killer cells (16), is also expressed on SCLC authors and are not to be construed as official or reflecting the views of the cells and other benign and malignant NE and nerve cells, Department of the Navy, the Department of Health and Human Services, or the although the precise antigenic determinant of the antibody has Department of Defense. 2To whom requests for reprints should be addressed, at NCI-Navy MOB, yet to be determined (17, 18). CgA was originally isolated from Naval Hospital. Building 8, Room 5101. Bethesda. MD 20814. the chromaffin granules of the adrenal medulla (19) and has 3The abbreviations used are: NE. neuroendocrine; SCLC-C, classic small cell lung cancer; SCLC-V, variant small cell lung cancer; NSCLC, non-small cell lung been found to be expressed by a variety of normal and neoplastic cancer; ExPuSC, extrapulmonary small cell carcinoma; Syn, Synaptophysin; CgA, human polypeptide hormone-producing tissues (20-22). Al chromogranin A; dopa, dihydroxyphenylalanine: DDC. L-dopa decarboxylase; though its function is not known, CgA is associated with the NSE, neuron-specific enolase; PAGE, polyacrylamide gel electrophoresis; PBS. phosphate-buffered saline; SCLC, small cell lung cancer: SDS, sodium dodecyl co-secretion of a wide variety of hormones, suggesting some sulfate. role in the secretory process (22). Because SCLCs have a paucity 6068
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. SYNAPTOPHYSIN IN LUNG CANCER CELL LINES of secretory granules (4), the use of CgA as a general NE marker h. DDC values of >4 units/mg of soluble protein were scored positive, may be limited only to those neoplasms containing more abun since minimal DDC activity can occur in most tissues, including normal dant dense core granules (5, 10, 23). These facts demonstrate lung. the need for additional NE markers with greater sensitivity and Western Blot Analysis. Tumor cell lines were evaluated by Western blot analysis for expression of Syn, using a modified method of the specificity. Towbin and Gordon technique (34, 35). Whole-cell lysates were ad Syn was originally isolated from bovine neurons and is a M, justed to equal protein concentrations and applied to the gel at 10-20 38,000 membrane-integrated glycosylated polypeptide to which Mgof protein/well. SDS-PAGE was performed under reduced condi a monoclonal antibody (SY 38) has been raised (24). Syn has tions and fractionated products were electroblotted onto nitrocellulose been demonstrated in a variety of normal and neoplastic NE membranes. The membranes were then treated with anti-Syn antiserum cells of neural and epithelial type (25, 26) and has been localized (SY38, 0.1 mg/ml) at a dilution of 1:100, followed by exposure to within normal and neoplastic cells of the bronchopulmonary rabbit anti-mouse IgG (lot 17529; Cappel Laboratories, Inc., Cochran- tract (27). While some results suggest that Syn immunoreactiv- ville, PA) at a dilution of 1:1000. Selective antibody deposition was ity is maintained in formalin-fixed paraffin-embedded speci identified with labeled '"I-Protein A. To confirm the specificity and mens (26-28), others have reported variable immunoreactivity competency of our immunostaining technique with the anti-Syn re agent, a parallel experiment was performed on identical cell lines, using (29). Our goal in the present study was to define the sensitivity an indifferent isotypically matched monoclonal antibody, MOPC 21, and specificity of Syn as a marker for NE differentiation in at a concentration of 0.1 mg/ml. lung cancer cell lines and to compare it to other general NE markers. RESULTS
MATERIALS AND METHODS The results for Syn staining distribution are summarized in Cell Lines. Four bronchial carcinoid, 4 ExPuSC, 10 SCLC-C, 7 Table 1. All carcinoids were positive for Syn and 3 of the 4 cell SCLC-V, 11 NSCLC, and 7 NSCLC-NE cell lines were established, lines had over 50% cells positive. Syn staining in small cell maintained, and characterized in our laboratory, as described previously carcinoma cells lines was more heterogeneous, with 14 of the (4, 5, 30, 31). Cell lines demonstrating substrate adherence were tryp- 21 cell lines positive. The 5 Syn-positive NSCLC cell lines all sinized by adding 10 ml of warm PBS to the flask and 5 ml of 0.05% belonged to the NSCLC-NE subtype. trypsin to stop cell adherence. Once trypsinization was complete (5-10 Western blot analysis was performed to demonstrate antibody min), 10 ml of fresh serum-containing medium were added to neutralize specificity on representative cell lines including 1 carcinoid, 1 the trypsin. The cell suspension was centrifuged at 1200 rpm for 5 min SCLC-C, 2 SCLC-V, and 1 ExPuSC (Fig. 1). Physical charac and resuspended in fresh medium. Trypsinized cells were allowed to terization of these cell lines by SDS-PAGE revealed a distinct recover for 2-3 h prior to immunocytochemical staining. All cell lines were washed twice in 10 ml of ice-cold PBS and cytocentrifuged onto cellular component which expressed Syn immunoreactivity. poly(L-lysine) (Sigma Chemical Company, St. Louis, MO)-coated This component migrated as a single band in Fig. 1, lanes I and 2, and as a series of bands in Fig. 1, lanes 3-5, with a slides. Immunocytochemical Staining. Immunocytochemical staining was determined molecular weight ranging from 43,000 to 45,000 performed by the avidin-biotin-peroxidase technique, using Vectastain under the reduced conditions. The reactivity was specific for ABC staining kits (Vector Laboratories, Burlingame, CA), following antibody SY38, since no immunoreactive bands were observed the vendor's instructions with a few modificai ions (10). Mouse mono in any of the cell lines when indifferent mouse monoclonal clonal anti-Syn (SY 38, lot 10644532-01) was purchased from Boehrin- antibody (MOPC 21) was substituted for SY38 (Fig. 1). ger Mannheim (Indianapolis, IN) and used at a 1:10 dilution. Mouse Syn, CgA, and DDC expression in all cell lines is summarized monoclonal anti-CgA (LK2H10) was a gift from Dr. Barry S. Wilson in Table 2. Syn was expressed more commonly in carcinoids (University of Michigan Medical Center, Ann Arbor. MI) and was used (100%) and SCLC (65%) than CgA and DDC. Four of 7 SCLC- at a dilution of 1:100. Immunoreactivity for Syn and CgA was tested following the fixation V were positive for Syn, while only 2 of 7 were positive for of freshly made cytospin preparations in methanol, acetone, and 95% CgA. In NSCLC cell lines the expression of Syn as well as the ethanol. Fixation for 10 min in ice-cold 95% ethanol appeared to give two other markers were restricted to the NSCLC-NE subtype, the best results (i.e., strongest staining with least background and good which showed average DDC levels of 273 ±43 units/mg. As preservation of morphology). Furthermore, pelleted cells were fixed for expected, SCLC-C and carcinoid cell types showed the highest l h at 4°Cin freshly prepared 4% neutral buffered paraformaldehyde solution and rinsed with ice-cold PBS. The cell pellets were embedded Table 1 Expression of synaptophysin staining distribution in all cell types in paraffin, and 5-^m sections were mounted on gelatin-coated slides. The distribution score for synaptophysin staining in each cell line ranges from Cytopreparations of each cell line were stained with hematoxylin and Oto 3;0 = no positive cells, 1 =
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ANTI-SYNAPTOPHYSIN INDIFFERENT MONOCLONAL MONOCLONAL ANTIBODY ANTIBODY Fig. l. Western blot analysis for Syn of ExPuSC (NCI-H510). SCLC-C (NC1- H146). SCLC-V (NCI-H446 and NC1-N417). and carcinoid cell line (NCT-H727). Whole cell Usâteswere subjected lo SDS-PAGE fractionation under reducing conditions and electroblotted to nitrocellulose membranes. The membranes were treated with antisynaptophysin anliserum (SY38. O.I mg/ml) at a dilution of 1:100. All cell lines contained peptides detected between a range of M, 43.000 to 45.000. When membranes were treated with undiluted indifferent monoclonal ; antibody MOPC 21 (0.1 mg/ml). no bands were seen, verifying the specificity of the immune reaction. T Table 2 Expression of synaptophysin. chromogranin, and i.-liOPA Jecarboxylase * Expression no. of cell lines (percentage) (no.)CarcinoidCell line (4)Small (75)10(48) (75)10(48)
cell carcinoma (21) SCLC-C (10) 7(70) 5(50) 8(80) SCLC-V (7) 4(57) 2(29) 0(0) ExPuSC(4)Non-small 3(75)5(28) 3(75)5(28) 2(50)6
cell lung cancer (18) (33) NSCLC-NE (7) 5(71) 5(71) 6(86) NSCLC. other (11)Syn"4(100)14(67)0(0)CgA"3 0(0)DDC*3 1 (9)
Total (43) 23(54) 18(42) 19(44)
AllNeuroendocrine''(28) lung carcinoma cell lines 20(71) 15(54) 17(61) Nonneuroendocrine' (II) 0(0) 0 (0) 1 (9) Total (39) 20(51) 15(38) 18(46) " Staining results of all cell lines were scored on cell distribution (0-3) and staining intensity (0-3). The sum of these values (0-6) was used to tabulate staining results for each antibody. Values of >3 were scored positive. Numbers in parentheses, percentage of cell lines scored positive. * One unit of enzyme activity is defined as I nmol MCO2/h. DDC values of >4 units/mg soluble protein were scored positive. Numbers in parentheses, percent B age of cell lines scored positive. ' NE lung cancer cell lines include carcinoid, SCLC-C, SCLC-V, and NSCLC- NE. Non-NE cell lines include other NSCLC cell lines only. Fig. 2. Photomicrographs of immunocytochemical staining in a bronchial carcinoid cell line. Immunocytochemical expression of synaptophysin and chro- DDC activity, 437 ±46 (mean ±SE) and 436 ±112 units/mg, mogranin A in bronchial carcinoid cell line NCI-H727. A, immunolocalization of respectively, while none of the SCLC-V showed any DDC synaptophysin using antibody SY 38. Note the diffuse cytoplasmic staining pattern and negative nuclei, x 900. A. the same cell line, demonstrating intense activity. chromogranin A immunoreactivity using antibody LK2H10. x 360. The staining The staining for both Syn and CgA was cytoplasmic, with no patlern of chromogranin A is strong and granular (insert, x 1800). staining in nuclei (Figs. 2-4). The Syn ¡mmunoreactivity was more diffuse and less intense than that of CgA and this differ ence was most evident in carcinoids (Fig. 2). Similar but con immunoreactivity in 11 of 14 (79%) and 8 of 13 (62%) cell siderably weaker staining was observed in paraffin-embedded lines, respectively. cell line pellets, which demonstrated a loss of Syn and CgA The concordance for Syn, CgA, and DDC expression in all 6070
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. SYNAPTOPHYSIN IN LUNG CANCER CELL LINES
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B B Fig. 3. Photomicrographs of immunocvlochcmical staining•••c-^i in SC LC <~ cell n lines.i- .. Fig...... 4. Photomicrographs , .. , , ' , of immunocvtochemical , . slamine uri. in a x,c-,~iNSCLC-NE /- v.r- 6. . . . ' . . . , ,.., ,„. ., C-/-I/-/~ ¡i cell line. The relatively abundant cvloplasm in scattered cells of Ihc NSC LC-NE A.,;'H-,_.,.,., positive, mostly J cvtoplasmic. „"„„„.. mimunostaining , with Si 3«in...... the SC LC-C cellcell .... line *,,-,NCI-HI570 U««<«A¿--r is positive for svnaptophvsini.-/ (A and ji. chromoeranin-./»>x A (B). line NCI-H345. x. 900. B, positive cvtoplasmic immunostainmg in the variant „.. SCLC cell line NCI-N417. using SV 38. This cell line lacked both CgA immu- noreactivitv and DDC activity hut was positive for Syn also in the Western blot analysis. Note the presence of nucleoli (arrows), characteristic of SCLC-V. X 110°- cell lines, 57% demonstrated concordant expression of Syn and CgA, while only 43% demonstrated concordance between Syn and DDC. Syn appears to be a more sensitive marker for SCLC- cell lines was statistically significant using Fisher's exact test V, which characteristically shows only partial expression of the (P < 0.05) (Table 3). Good concordance was observed for all NE phenotype and is associated with an aggressive clinical subtypes except SCLC-V. Of the 57% Syn-positive SCLC-V course. 6071 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. SYNAPTOPHYSIN IN LUNG CANCER CELL LINES Table 3 Concordant expression ofsynaptophysin, chromogranin A, and ¡.-dopa tant, since it has been suggested that NSCLC-NEs are initially decarboxylase Concordance is when both markers are present in a particular cell line. The more sensitive to chemotherapy than other NSCLCs (11, 36). numbers in parentheses represent percentage. Concordances were statistically A marked loss of Syn immunoreactivity was demonstrated in significant (P < 0.05) for all cell types (n = 43) using Fisher's exact test. 11 of 14 (79%) of the formalin-fixed paraffin-embedded cell Expression pellets (previously showing immunoreactivity in freshly pre no. of cell lines (percentage) pared ethanol-fixed specimens). These results support previ ously reported findings that various fixation techniques can (no.)CarcinoidCell line andCgA°3(75)14andDDC*3(75)13(62)andDDC*3 alter the immunocytochemical demonstrability of Syn (29). (4)Small (75)14(67) Moreover, it appears that cultured cells are even more likely to cell carcinoma (21) (67) lose their immunoreactivity due to formalin fixation and par SCLC-C(IO) 6(60) 7(70) 6(60) SCLC-V (7) 4(57) 3(43) 5(71) affin embedding than routinely processed tissue samples col ExPuSC(4)Non-small 4(100)16(89) 3(75)17(94) 3(75)17(94) lected in the practice of surgical pathology (28). cell lung cancer (18) Our study on cell lines is in accordance with previously NSCLC-NE (7) 5(71) 6(86) 6(86) published results showing that immunoreactive Syn is present (11)TotalNSCLC, other 11(100)33 11(100)33 11(100)35(81) in vivo in most but not all NE tumors of the lung (25-28, 37, (43)Syn" (77)Syn" (77)CgA" 38). While many studies have included only a few cases, Gould " Staining results for all cell lines were scored on cell distribution (0-3) and et al. (25) in their study on 270 tumors reported that up to 17 staining intensity (0-3). The sum of these values (0-6) was tabulated for each of 19 (88%) pulmonary carcinoids and 6 of 14 (43%) NE antibody. Values of >3 were scored positive. * One unit of enzyme activity is defined as 1 nmol l4CO2/h. DDC values of >4 carcinomas of the lung were positive for Syn. Higher incidences units/mg soluble protein were scored positive. were reported for lung NE carcinomas (88%) by Lee et al. (27) and for SCLC (79%) by Kayser et al. (28), who also noted that DISCUSSION 8 of 74 (10%) NSCLC were positive for Syn. In our in vitro study, 70% of SCLC-C and 67% of all small cell carcinoma cell Our findings indicate that Syn is a specific and sensitive immunocytochemical marker for NE differentiation in lung lines tested positive for Syn. In summary, these studies suggest cancer cell lines and ExPuSC. We demonstrated that Syn that, while most lung NE tumors contain Syn, some tumors do immunoreactivity is expressed more often in bronchial carci- not express this protein. Presently, the biological or clinical noid, SCLC-C, and SCLC-V cell lines, in comparison to im implications of this are not fully understood. munoreactivity with CgA. These results suggest that Syn may The gene for human Syn has been previously cloned and was be a more sensitive biochemical marker for NE differentiation demonstrated to generate a single mRNA transcript which than CgA in these subtypes. Antibodies to both Syn and CgA encodes a 30,700-amino acid precursor protein (39). Although showed positivity with the same frequency in ExPuSC and the deduced molecular weight of this prepro-Syn molecule is NSCLC-NE cell lines, while none of the other NSCLC cell 33,312, multiple forms have been reported in a variety of human lines expressed either Syn or CgA. tissue, ranging in molecular weight from 38,000 to 40,000 (39). When all cell types were considered, the concordant expres This reported heterogeneity in the calculated molecular weight sion of two of the three NE markers (Syn, CgA, DDC) was of the Syn precursor, particularly evident in tumor samples, has highest for CgA and DDC (81%). These results are in agreement been attributed to the degree of glycosylation obtained during with our earlier findings, which report excellent concordance posttranslational modification. Studies by Navone et al. (40) between CgA and DDC in both SCLC and NSCLC lung tumors and Rehm et al. (41) have shown that chemical deglycosylation (3). Syn was expressed in 70% of the SCLC-C cell lines, which or inhibition of de novo glycosylation renders the Syn precursor correlates well with a previous study on tumor specimens (24). of «34,000. Our biochemical characterization of Syn-immu- Of the SCLC-V cell lines which are characterized by altered noreactive proteins in lung tumor cell lines revealed a slightly morphology and partial lack of the NE program (4, 5), 4 of 7 higher molecular weight form, ranging from 43,000 to 45,000. (57%) expressed Syn, while only 2 of 7 (29%) stained positively This is in accordance with the slightly lower electrophoretic for CgA. The presence of Syn in SCLC-V was also confirmed mobility that has been reported for selected tumors, including by Western blot analysis. None of the SCLC-V cell lines were a bronchial carcinoid. paragangliomas, and retinoblastomas found to express DDC. This is in agreement with previous (26, 37, 42). Interestingly, the SCLC-C (NCI-HI46) and Ex findings which report a loss of dense core granules and lack of PuSC (NCI-H510) cell lines expressed a higher molecular DDC activity in this SCLC subset (4, 5). Because patients with weight form of M, 45,000, when compared to the SCLC-V SCLC-V may respond less well to chemotherapy (6), the detec (NCI-H446 and NCI-N417) and carcinoid (NCI-H727) cell tion of these tumors is of clinical and biological interest. lines, in which a M, 43,000 product was noted. These data We have recently shown that as many as 20% of NSCLC suggest that, in addition to extensive glycosylation of the Syn tumors, most commonly adenocarcinomas and large cell carci nomas, contain multiple NE markers (NSCLC-NE) (10). Pre molecule, there may be tumor selectivity associated with this liminary results suggest that patients with NSCLC-NE tumors posttranslational modification step in lung and extrapulmonary may respond more favorably to chemotherapy than patients tumor cell lines. with other NSCLC tumors (11). We included a panel of 7 The function of Syn is not known. Syn is a hexameric protein NSCLC-NE cell lines in the present study and found that 4 of capable of forming transmembrane channels (43) and is one of 7 (57%) NSCLC-NE cell lines expressed all three NE markers, the major calcium-binding proteins of the synaptic vesicle mem Syn, CgA, and DDC. Of interest, 2 of these were large cell brane, with the binding site on its cytoplasmic domain (41). carcinoma cell lines, 1 bronchioloalveolar and 1 adenosqua- Moreover, Syn is one of the major target proteins of endoge mous carcinoma. Thus, the addition of Syn as a marker for NE nous tyrosine phosphorylation in highly purified synaptic vesi differentiation in NSCLC tumors diagnosed by conventional cles, as well as in synaptosomes from rat forebrain (44). It can light microscopy techniques may prove to be clinically impor also be phosphorylated by the c-src-encoded protein tyrosine 6072
Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. SYNAPTOPHYSIN IN LUNG CANCER CELL LINES kinase.4 While Syn was initially localized in intracytoplasmic nervous tissue. Nature (Lond.). 306: 179-181. 1983. 19. O'Connor, D. T.. Frigon, R. P.. and Sokoloff. R. L. Human chromogranin clear vesicles, subsequently smaller amounts were also found in A: purification and characterization from catecholamine storage vesicles of large dense core vesicles (40, 45-49). Thus, it can be speculated human pheochromocytoma. Hypertension (Dallas). 6: 2-12. 1984. O'Connor, D. T., Burton. D.. and Deftos. L. J. Immunoreactive human that Syn has a role in the intracellular vesicle transport system, 20. chromogranin A in diverse polypeptide hormone producing human tumors in communication with membrane and cytoskeleton, or in and normal endocrine tissues. J. Clin. Endocrinol. Metab.. 57: 1084-1086. 1983. exocytosis. In cancer cells, this may involve intracellular traf O'Connor. D. T.. Burton. D., and Deftos. L. J. Chromogranin A: immuno- 21. ficking of growth factors and oncogenes. histology reveals its universal occurrence in normal polypeptide hormone In summary, we recommend that Syn be added to the panel producing endocrine glands. Life Sci.. 33: 1657-1663, 1983. 22. Deftos, L. J., Linnoila, R. !.. Carney, D. N.. Burton. D. W., Leong. S. S., of general NE markers currently in use for the following rea O'Connor. D. T., Murray, S. S., and Gazdar. A. F. Demonstration of sons: (a) Syn may be a more specific and sensitive biochemical chromogranin A in human neuroendocrine cell lines by immunohistology marker for NE differentiation than CgA in carcinoid and classic and immunoassay. Cancer (Phila.). 62: 92-97, 1988. SCLC cell types; (b) Syn is a more sensitive marker for NE 23. Wilson. B. S., and Lloyd, R. V. Detection of chromogranin in neuroendocrine neoplasms which lack part of the NE program (i.e., SCLC-V); cells with a monoclonal antibody. Am. J. Pathol.. 115: 458-468. 1984. 24. Wiedenmann. B.. and Franke, W. W. Identification and localization of (c) Syn may be helpful in identifying differentiating, and clas synaptophysin. an integral membrane protein of M, 38.000 characteristic of sifying small cell carcinomas of extrapulmonary or unknown presynaptic vesicles. Cell. 41: 1017-1028. 1985. 25. Gould. V. E., Wiedenmann. B., Lee, I., Schwechheimer. K., Dockhorn- origin; and (epithelium. Biochim. Biophys. Acta, 541: 415-419, 1978. cinoma compared to "pure" small cell subtypes. Cancer (Phila.). 50: 2894- 33. Beaven. M. A., Wilcox, G.. and Terpstra. G. K. A microprocedure for the measurement of I4CO¡releasefrom ["Cjcarboxyl labelled amino acids. Anal. 2902. 1982. Biochem.. «4:638-641. 1978. 7. Ibrahim. N. B. N., Briggs, J. C.. and Corbishley, C. M. Extrapulmonary oat Towbin, H., and Gordon. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. A Comparison of Synaptophysin, Chromogranin, and l-Dopa Decarboxylase as Markers for Neuroendocrine Differentiation in Lung Cancer Cell Lines
Sandra M. Jensen, Adi F. Gazdar, Frank Cuttitta, et al.
Cancer Res 1990;50:6068-6074.
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