Coexpression of Cholesterol Sulfate and Cytokeratin As Tumor Markers in Well-Differentiated Squamous Cell Carcinoma of the Human Uterine Cervix

Vol. 4, 2985-2990, December 1998 Clinical Cancer Research 2985

Coexpression of Cholesterol Sulfate and Cytokeratin as Tumor Markers in Well-Differentiated Squamous Cell Carcinoma of the Human Uterine Cervix

Kazushige Kiguchi,’ Masao Iwamori, INTRODUCTION Shizuka Yamanouchi, Isamu Ishiwata, It is well known that the pattern of expression of glyco- Masahiko Saga, and Akira Amemiya conjugates on the cell membrane changes during malignant transformation, cellular differentiation, and proliferation ( 1) and Department of Obstetrics and Gynecology, Toyoko Hospital, St. can be useful markers for the diagnosis of several tumors (2-5). Marianna University School of Medicine, Kanagawa 21 1 [K. K., S. Y., M. S.]; Department of Biochemistry, Faculty of Medicine, However, glycoconjugates, like tumor-associated antigens, are University of Tokyo, Tokyo 113 [M. I.]; Ishiwata Obstetrics and generally species specific in nature like the blood group antigens Gynecologic Hospital, Ibaraki-ken 310 [I. I.]; and Department of and are consequently not applicable for the characterization of Obstetrics and Gynecology, St. Marianna University School of cell type-specific alterations commonly observed during cellular Medicine, Kanagawa 216 [A. A.], Japan differentiation in different animal species. Among membrane constituents, the pattern of distribution of CS2 and CSE has been

ABSTRACT shown to be similar among tissues and cells of several mammalian species and are thought to be involved in the common The expression of cholesterol sulfate (CS) is known to functions of individual tissues (6). In fact, CS, which was increase during squamous differentiation of keratinocytes present in squamous epithelial cells, was shown to be a key and to activate the #{128},i, and forms of protein kinase C as molecule involved in keratinocyte differentiation through the a signal transduction molecule for the subsequent expression activation of the e, ‘ri, and isoforms of PKC (7, 8). The of transglutaminase-1 (TG-1) and cytokeratins. To gain fur- substrate for PKC’q has been shown to be TG-l , the activity of ther insight into the regulation of cellular differentiation and which is thought to be positively or negatively regulated by tumorigenesis by CS, we examined the concentration and phosphorylation with PKC’q, and which is a marker of cellular the potential for synthesis of CS in seven and four surgical differentiation (7, 9). In addition, cholesterol sulfotransferase, in specimens from human ovarian and uterine cervical cancer the first step of the unique signal transduction system, was patients, respectively, and eight cell lines established from shown to be activated in the uterine endometrium of pseudo- human uterine cervical cancer patients and compared them pregnant rabbit induced with estrogen and chorionic gonado- for the rate of expression of cytokeratin. CS was present in tropin (10) and in rnurine skin treated with phorbol ester (1 1), all of the uterine cervical cancer tissue specimens but only in indicating that the expression of CS is regulated by differenti- the mucinous type of cystadenocarcinoma among ovarian ation- and proliferation-related signals. A similar regulation of cancer tissue specimens, and cytokeratin was highly ex- sulfolipid expression by steroids was observed for CSE in the pressed in the tissues with a high concentration of CS, which endometrium of a human uterus in the luteal phase ( 1 2). Thus, were classified as well-differentiated on the basis of morpho- the sulfated lipids, CS and CSE, are expressed in relation to the logical examination. Similarly, cells derived from a keratin- cellular functions during differentiation and could be useful izing type of well-differentiated cervical carcinoma demon- markers for the process of tumorigenesis. To assess the possible strated strong potential for synthesis of CS, stained positive involvement of sulfolipids in the biological properties of cancer with anti-cytokeratin antibody, and exhibited a higher spe- cells, we compared the concentrations of sulfolipids relative to cific activity of TG-1, whereas the cells without CS did not the expression of cytokeratins in the tissues and cells of various stain positive with anti-cytokeratin antibody and exhibited a gynecological cancers. lower specific activity of TG-1. These findings indicate that CS is coexpressed with TG-1 and cytokeratin in the well- MATERIALS AND METHODS differentiated types of squamous cell cancers as a tumor Materials. Murine monoclonal antibodies, K8. 12, di- marker. rected against cytokeratin 13, and K8.60, directed against cytokeratins 1, 10, and I 1, were purchased from Sigma Chemical Co. (St. Louis, MO). CS was synthesized by sulfation of the 33-hydroxy group of cholesterol (Wako Chemicals, Tokyo, Received 3/13/98; revised 8/31/98; accepted 9/14/98. Japan) with pyridine sulfate. The CSE and GMI were prepared The costs of publication of this article were defrayed in part by the from human brain and placenta, respectively, in our laboratory. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 1 8 U.S.C. Section 1734 solely to indicate this fact. C To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, Toyoko Hospital, St. Marianna University 2 The abbreviations used are: CS, cholesterol sulfate: CSE, Lactosylce-

School of Medicine, 3-435 Kosugi, Nakahara-ku, Kawasaki, Kanagawa ramide-1I3-sulfate; PKC, protein kinase C; TG-l , transglutaminase- I; 211, Japan. Phone: 81-44-722-2121; Fax: 81-44-722-2738. GMI, II3NeuAc-LacCer.

tions by DEAE-Sephadex A-25 (acetate form) column chroma-

tography; the acidic lipids were then eluted with 0.3 M sodium . Cs acetate in methanol. The ester-containing lipids in the acidic lipid fraction were removed by mild alkaline hydrolysis, fol- S CSE lowed by dialysis. The acidic lipids thus obtained were devel- #{149}0 oped on TLC plates with chloroform:methanol:0.5% CaCl2 in water (55:45: 10, v/v) and chloroform:methanol:acetone:acetic acid:water (8:2:4:2:1, v/v). Orcinol-H2SO4 reagent was used for * #{149}s#{149} GM3 visualizing glycolipids, and cupric acetate-phosphoric acid rea- - gent was used for organic compounds. 1 2 3 4 5 6 7 8 9 10 11 Quantitative Determination of CS and CSE. The concentrations of acidic lipids were determined densitometrically at Fig. 1 TLC of acidic lipids and concentrations of CS and CSE in the cancer tissue specimens from patients with ovarian and uterine cervical 500 nm with a dual-wavelength TLC densitometer (CS-9000; carcinomas. The acidic lipids, corresponding to 2 mg of dry tissue Shimadzu Co., Kyoto, Japan) after locating the spots with cupric weight, were developed with chloroform:methanol:acetone:acetic acid: acetate-phosphoric acid or orcinol-H2SO4 reagent as stated water (8:2:4:2: 1 , v/v), and the spots were visualized by spraying with above. Known amounts of chemically synthetic CS and CSE cupric acetate-phosphoric acid reagent and heating the plate at 230#{176}C. from human brain were spotted on the same plates for the Lanes 1-4, serous cystadenocarcinoma of ovary; Lanes 5 and 6, mucinous cystadenocarcinoma of ovary: Lane 7, clear cell carcinoma of preparation of standard curves, which were linear from 0. 1 p.g to ovary: Lanes 8-10. nonkeratinizing large cell type of uterine cervical 1 p.g of CS and CSE, respectively. carcinoma: !Lflie I 1, nonkeratinizing small cell type of uterine cervical Immunohistochemical Staining. The 4-p.m sections of carcinoma. GM3. GMI. formalin-fixed human ovarian and uterine cervical cancer tissue specimens and cervical cancer-derived cells, of which the en- dogenous peroxidase was inactivated by preincubation with 0.3% H2O2 in methanol, were incubated with monoclonal anti- Tumor Tissues. Tumor tissues were obtained from the cytokeratin antibodies K8. 12 and K8.60 at 4#{176}Covernight and Department of Obstetrics and Gynecology, Toyoko Hospital, St. subsequently treated with biotinylated goat anti-mouse 1gM Marianna University School of Medicine. Histological classifi- antibody and peroxidase-conjugated avidin (Vector Laborato- cation of the specimens was performed according to the criteria ries, Inc., Burlingame, CA) at 37#{176}Cfor I h, followed by reaction defined by the International Federation of Gynecology and in a solution of 3,3-diaminobenzidine tetrahydrochloride and Obstetrics. The specimens comprised seven ovarian carcinomas H202 in 50 mM Tris-HCI buffer (pH 7.4) at room temperature (four serous papillary cystadenocarcinomas, two mucinous cys- for 10 mm. The nucleus was stained with hematoxylin. tadenocarcinornas, and one clear cell carcinoma) and four uter- Incorporation of Radioactive Sulfate into Sulfolipids. me cervical carcinomas (three large cell nonkeratinizing types and one small cell nonkeratinizing type). The samples were To examine the potential for synthesis of sulfolipids, the eight immediately stored at -70#{176}C until use. cell lines from uterine cervical carcinomas were cultured in a Cell Lines. Eight cell lines, established from squamous medium containing 3.7 kBq of Na235SO4 (3.7 GBq/mmol; Am- cell carcinoma of the uterine cervix, were used for this experi- ersham, Buckingharnshire, United Kingdom) for 20 h, and the ment. The cell lines consisted of two keratinizing types (TCS cells were collected with a scraper. Lipids were extracted from and HHUS), three large cell non-keratinizing types (HKTUS, the cell pellet as described above, and an aliquot of the extracts HKMUS, and 5KG-Il), and two small cell nonkeratinizing types was applied on the TLC plate, which was developed with of squamous cell carcinomas (QGU, AMCC-l, and HKUS). The chloroform:methanol:acetone:acetic acid:water (8:2:4:2:1, v/v), cell lines HHUS, HKTUS, HKMUS, 5KG-I!, and HKUS were and the radioactivity incorporated into CS and CSE was meas- established by Dr. Isamu Ishiwata (Ishiwata Obstetrics and ured using an image analyzer (BAS-2000; Fuji, Tokyo, Japan). Gynecological Hospital), and the cell lines TCS, QG-U, and Assay of TG-1. For determination of TG-l activity, the AMCC-l were kindly provided by Prof. Rikuichi Izurni human uterine cervical carcinoma-derived cells were homoge- (Toyama Medical and Pharmaceutical University, Faculty of nized in 50 mM Tris-HC1 (pH 7.5) containing 0.25 M sucrose and Medicine, Toyarna, Japan), Dr. Harukazu Mashiba (National 1 ms EDTA. The homogenates were centrifuged at 30,000 X g Kyushu Cancer Center, Fukuoka, Japan), and Dr. Hirornitsu for 30 mm, and the resultant supernatant was used as the enzyme Yabushita (Aichi Medical University, Aichi, Japan), respec- source. The assay mixtures comprised 50 mrvi Tris-HC1 (pH 8.0), tively. The cells were grown in Ham’s F-12 medium supple- 10 msi CaC12, 5 mM DTT, 0.6 mg of dimethyl casein, [‘4C]- mented with 10% FCS. putrescine (1.67 kBq, 2.96 GBq/mmol; DuPont NEN, Boston, Preparation of Sulfolipids. The extraction and prepara- MA), and enzyme in a total volume of 0.6 ml. After incubation tion of crude lipids from gynecological cancer tissues were at 37#{176}Cfor 1 h, the reaction was terminated by the addition of carried out according to the methods reported previously (13). 0.6 ml of 10% trichloroacetic acid, followed by dilution with 5 The extracts were mixed, and then the lipid-bound phosphorus ml of 5% trichloroacetic acid containing 0. 1 % cold putrescine. and cholesterol content in the total lipid extract was determined The solution was then filtered through Whatman GF/A filter by Bartlett’s method (14) and by gas-liquid chromatography discs, which were washed with 10 ml of 5% trichloroacetic acid with 5a-cholestane as an internal standard (15), respectively. containing 0. 1% cold putrescine, followed by washing with 10 The total lipids were fractionated into neutral and acidic frac- ml of ethanol. The radioactivity on the discs was counted with

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Fig. 2 Immunohistochemical staining with antibody K8.60 in the cancer tissue specimens from patients with ovarian and uterine cervical carcinomas. A, serous cystadenocarcinoma of ovary; B, mucinous cystadenocarcinoma of ovary; C. nonkeratinizing large cell type of uterine cervical carcinoma: D, nonkeratinizing small cell type of uterine cervical carcinoma.

a liquid scintillation counter (TriCarb 1500; Packard, Orange- immunohistochemical staining with anticytokeratin antibodies vale, CA; Ref. 16). K8. 12 and K8.60, and the staining patterns were compared according to the intensity and incidence of staining as shown in

RESULTS Fig. 2. No positive reaction was observed on staining with Sulfolipids in the Ovarian and Uterine Cervical Cancer antibody K8. 12, whereas antibody K8.60, which reacts with Tissues. Although sialoglycolipid GM3 was contained in all of cytokeratins 1, 10, and 1 1, elicited a positive reaction in tissues the tissues examined, the level of expression of sulfolipids was other than those of clear cell carcinoma of the ovary (Fig. 2). different among the cancer tissue specimens from patients with Mild to moderately positive staining was observed in one of the ovarian and uterine cervical cancers (Fig. 1). In the case of four specimens of ovarian serous cystadenocarcinomas and one ovarian cancers, CSE was not detected in the specimens of of the two specimens of ovarian mucinous cystadenocarcino- serous cystadenocarcinoma, whereas it was the major compo- mas. On the other hand, a moderate to strongly positive reaction nent of the acidic lipid fraction in those of mucinous cystade- was observed in the specimens from the patients with uterine nocarcinoma, and only faint expression was observed in the cervical squamous cell carcinoma, in two of the three specimens specimens of clear cell carcinoma. In the case of uterine cervical of large cell nonkeratinizing type of carcinoma, and the one cancer specimens, CSE was not detected except in the speci- specimen of the small cell nonkeratinizing type, all of which mens of the large cell nonkeratinizing type. On the other hand, were classified as well-differentiated on the basis of morpho- CS was contained in all of the ovarian mucinous cystadenocar- logical examination (Table 1). The tissues that stained strongly cinoma and cervical squamous cell carcinoma specimens and in with anticytokeratin antibody expressed CS in relatively high one of the four specimens of ovarian serous cystadenocarci- concentrations, indicating coexpression of CS and cytokeratin. noma. A significantly high concentration of CS was observed in Correlation between the Potential for Synthesis of CS the specimen of small cell nonkeratinizing type of cervical and Expression of Cytokeratin in Cell Lines from Human carcinoma. Thus, CS was commonly expressed in cervical squa- Uterine Cervical Carcinomas. To further confirm the obser- mous cell carcinoma; both CS and CSE could be detected in vations in cancer tissue specimens, we analyzed the synthetic mucinous cystadenocarcinoma of the ovary, whereas CSE was activity and concentration of CS, specific activity of TG-l, and not detected in serous cystadenocarcinoma of the ovary (Fig. 1). expression of cytokeratin in eight cell lines established from Immunohistochemical Analysis Using Anticytokeratin human cervical carcinoma. As shown in Fig. 3A, when the cells Antibodies. Tissues obtained from the patients suffering from were cultured in a medium containing Na235SO4, an intense ovarian and uterine cervical carcinomas were examined by radioactive spot migrating to the position of CS was detected

Table I Concentrations of cholesterol sulfate and sulfatide and expression of cytokeratin in the cancer tissue s pecimens from patients with ovarian and uterine cervical carcinomas The results obtained in Fig. 2 were assessed according to the intensity and incidence of staining.

CS CSE Expression of Case Pathology (nmol/mg dry weight) cytokeratin” I Serous cystadenocarcinoma of the ovary’ ., -

:1 0.01 ++

5 Mucinous cystadenocarcinoma of the ovaryc 0.01 1 .2 - 6 0.02 0.8 +

7 Clear cell carcinoma of the ovary 0.01 - 8 Nonkeratinizing large cell carcinoma of the uterine cervix” 0.08 + + + + 9 0.01 ++++

10 0.01 - I 1 Small cell carcinoma of the uterine cervix 0.04 0.03 + + +

Incidence

Low Moderate High Intensity Weak + ++ ++ Moderate ++ +++ +++ Strong ++ +++ ++++ (1 _,negative staining. “Cases 1-4. ( Cases 5 and 6. “Cases 8-10.

only in the TCS cells and to a lesser extent in the QG-U cells, A but the other cell lines, other than the HKUS cells, in which 5s was incorporated into sulfated glycolipid did not possess the potential for synthesis of either of the sulfated lipids CSE or CS. Cs A TLC of the acidic lipids in the cell lines revealed that TCS cells contained CS at the concentration of 452.6 nmol/p.g of CSE protein, whereas the other cell lines, other than the QG-U cells, which contained 22 nmol/p.g of protein, did not contain CS even in trace amounts (Fig. 3B and Table 2). The expression of TG-l and cytokeratin in the TCS cells with CS and the 5KG-Il cells - without CS were compared relative to that of CS. As shown in -1 ;D. I U) Table 2 and Fig. 4, the specific activity ofTG-l in the TCS cells C) ;; ;c , c#{149}):1 C C) C was significantly higher than that in the other cells including () L. Cl) - Cl) 5KG-Il cells, and the staining of the TCS cells with antibody K8.60 was more intense than that of the 5KG-Il cells, indicating B coexpression of CS with the differentiation-associated markers TG- I and cytokeratin in uterine cervical carcinoma-derived cells.

DISCUSSION In this study, we demonstrated that the sulfated lipids CS and CSE could be useful markers for the characterization of human gynecological cancers. In the case of ovarian carcinomas, CSE was the major component of the acidic lipids in mucinous cystadenocarcinoma ( 1 3) and was detected to a lesser -I i.Q 0.5 0.2 extent in clear cell carcinoma, whereas it was not detected at all 9 -1 OC C CS in serous cystadenocarcinorna. Also, CS was present in all of the = specimens of mucinous cystadenocarcinoma and one of the four Fig. 3 A, autoradiogram of the TLC of the lipids in uterine cervical specimens of serous cystadenocarcinoma. In the case of uterine carcinoma-derived cells cultured in a medium containing Na,35SO4 (3.7 cervical carcinomas, CS was expressed in all of the cervical kBq) for 20 h. B, TLC of acidic lipids in uterine cervical carcinoma- squamous cancers examined, whereas only one specimen of the derived cells. TLC plates were chromatographed as in Fig. I.

Table 2 Synthetic potential and concentration of CS and activity of TG- 1 in cell lines derived from the squamous cell carcinoma of human uterine cervix

Incorporation of S in CS Concentration of CS Activity of TG- 1 Cell line (PSL#{176}/mgprotein) (nmol/p.g protein) (pmol/mg protein/30 mm)

5KG-lI 0 0 8

HKMUS 0 0 - HKTUS 0 0 12 QG-U 427 22.02 30

AMCC-l 0 0 - HKUS 0 0 25

HHUS 0 0 - TCS 5090 452.60 540 a PSL, photostimulat ed luminescence; - , not determined.

A by proteases from secretory glands. Mucinous cystadenocarci- % norna with a high concentration of CSE appeared to originate from cells having a function similar to those of cells in secretory glands and consequently qualified as a differentiation-associated s . antigen. Expression of CSE in human uterine endometrium is ; . e;#{149}-- :: #{149} known to be characteristic in the luteal phase, which is induced by estrogen and progesterone stimulation (12, 19), suggesting

,-. I-. that GalCer sulfotransferase, responsible for the synthesis of .: CSE, is regulated during differentiation of uterine epithelial ,,. 1 cells by steroid hormones. Uterine endometrial carcinoma-de- .‘#{176} 7. rived cells also expressed sulfated glycolipids as cancer-associ- r ated antigens at a significantly high frequency, indicating that sulfated glycolipids including CSE are primarily related to the phenotype of cancer cells in the uterus (20). The original cells of ovarian mucinous cystadenocarcinoma are probably similar to B those of uterine endometrial carcinoma with respect to their -‘ differentiation under the influence of steroid hormones. On the ) other hand, CS was reported to be a marker of epithelial differentiation in epidermal keratinocytes and tracheal epithelial cells, along with keratins, fillaggrin, involucrin, loricrin, TG-l , galac-

‘ tose-binding Mr 14,000 lectin and cornified envelopes (21, 22). Expression of CS in the uterine endometrium of rabbit was also : #{149} stimulated by the administration of steroid hormones (10), and that in the epidermal keratinocytes was enhanced by phorbol-

0 l2-myristate-13-acetate, tumor growth factor-3, and high Ca2, .;‘ #{149}0St all of which are chemical inducers of squamous differentiation (11, 21, 23). Because CS was clarified to modulate the activity of TO- 1, it possibly functions in the initial step of signal Fig. 4 Immunohistochemical staining with antibody K8.60 of uterine transduction for squamous differentiation (7, 8). In fact, choles- cervical carcinoma-derived cells TCA (A) and 5KG-Il (B). terol sulfotransferase for the synthesis of CS, which was not detected in murine skin at day 14 of gestation, was abruptly expressed in association with the formation of a multilayered large cell nonkeratinizing type revealed CSE expression. Thus, structure at day 16, followed by the expression of TG-l, corni- the concentrations of CSE and CS in the gynecological cancer fled envelopes, and large keratin bundles, suggesting the pro- tissues were different between ovarian and cervical squamous grammed expression of squamous differentiation-associated carcinomas and between mucinous and serous cystadenocarci- molecules containing CS (16). These findings indicate that CSE noma of the ovary. CSE was contained in relatively high con- and CS are probably related to the structures of the cell surfaces centrations in normal tissues, such as brain, kidney, and gastro- of glandular and squamous epithelial cells, respectively, and that intestinal tract (6), and was localized in the epithelial cell lining cervical squamous cell carcinoma cells follow these lines of of gastric mucosa (1 7). Because one of its major functions in the differentiation. Immunohistochemical analysis of cancer tissues gastrointestinal tract was shown to be the inhibition of several using anticytokeratin antibody revealed that cervical carcinomas digestive enzymes for the protection of epithelial cells from expressing CS exhibited moderate to strongly positive staining, proteolysis (18), its expression in cancer cells was thought to whereas the tissues without CS were weakly stained, indicating reflect the property of a tissue for protecting itself from autolysis the coexpression of both markers in gynecological cancers.

Moreover, among cell lines established from the cervical squa- sulfate due to up-regulation of cholesterol sulfotransferase and concur- mous carcinomas of human uterus, the TCS cells having a high rent down-regulation of cholesterol sulfate sulfatase in the uterine en- concentration of CS and a high CS-synthetic potential revealed dometria of rabbits. J. Biochem., 116: 657-662, 1994. high specific activity of TO- 1 and more intense expression of I 1. Yamamoto, S., Jiang, H., and Kato, R. Involvement of prostag- landin E2 in the tumor promoter phorbol ester caused increase in cytokeratin than the 5KG-Il cells not containing CS. Thus, epidermal cholesterol sulfotransferase activity. Carcinogenesis (Lond.), coexpression of CS with the differentiation-associated mole- 12: 1145-1147, 1991. cules TO- 1 and cytokeratin was also demonstrated in cancer 12. Kubushiro, K., Oijima, K., Mikami, M., Nozawa, S., lizuka, R., derived-cells, as in cancerous tissues. An experiment on the Iwamori, M., and Nagai, Y. Menstrual cycle-associated alteration of activation of TO-l with exogenous CS in relation to the forma- sulfogalactosyl ceramide in human uterine endometrium. Possible in- tion of a cornified envelope and expression of cytokeratin is duction of glycolipids sulfation by sex steroid hormones. Arch. Bio- now in progress in our laboratory to clarify the functional chem. Biophys., 268: 129-136, 1989. significance of CS during the process of carcinogenesis. 13. Kiguchi, K., Takamatsu, K., Tanaka, J., Nozawa, S., Iwamori, M., and Nagai, Y. Glycosphingolipids of various human ovarian tumors: a significantly high expression of I (3)SO3Ga1Cer and Lewis antigen in ACKNOWLEDGMENTS mucinous cystadenocarcinoma. Cancer Res., 52: 416-421, 1992. We thank Y. Koshitaka, St. Marianna University School of Med- 14. Bartlett, G. R. Phosphorous assay in column chromatography. icine, for help on the immunohistochemical studies Prof. R. Izumi, and J. Biol. Chem., 234: 466-468, 1959. Toyama Medical and Pharmaceutical University, Dr. H. Mashiba, Na- 15. Iwamori, M., and Moser, H. W. Above normal urinary excretion of tional Kyushu Cancer Center, and Dr. H. Yabushita, Aichi Medical urinary ceramides in Farber’s disease and characterization of their University, for providing cell lines. We also thank Dr. K. Kiguchi, M. D. components by high performance liquid chromatography. Clin. Chem., Anderson Cancer Research Center, Houston, TX, for helpful comments. 21: 725-729, 1975. 16. Kagehara, M., Tachi, M., Harii, K., and lamori, M. Programmed REFERENCES expression of cholesterol sulfotransferase and transglutaminase during 1. Hakomori, S. Glycosphingolipids as differentiation-dependent, tu- epidermal differentiation of murine skin development. Biochim. Bio- mor-associated markers and as regulators of cell proliferation. Trends phys. Acta, 1215: 183-189, 1994. Biol. Sci., 9: 453-458, 1984. 17. 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K Kiguchi, M Iwamori, S Yamanouchi, et al.

Clin Cancer Res 1998;4:2985-2990.

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