Transferrin receptor in oral tumors

Transferrin receptor in oral tumors

Int. ,L Oral Maxillofac. Surg. 1994; 23:430-433 Printed in Denmark. All rights reserved Copyright © Munksgaard 1994 [ntemationa]Joumalof Orctl& Maxi...

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Int. ,L Oral Maxillofac. Surg. 1994; 23:430-433 Printed in Denmark. All rights reserved

Copyright © Munksgaard 1994 [ntemationa]Joumalof

Orctl& Maxillofacial Surgery ISSN 0901-5027

Transferrin receptor in oral tumors

Takao Miyamoto 1, Nobuyuki Tanaka 1, Yoshinobu Eishi 2, Teruo Amagasa 1 1First Department of Oral and Maxillofaeial Surgery, Faculty of Dentistry, and 2Department of Pathology, School of Medicine, Tokyo Medical and Dental University, Japan

T. Miyamoto, N. Tanaka, Y. Eishi, T. Amagasa." Transferrin receptor in oral tumors. Int. J. Oral Maxillofac. Surg. 1994; 23: 430-433. © Munksgaard, 1994 Abstract. The transferrin receptor (TfR) appears in vigorously proliferating cells. We did an immunohistochemical study of TfR in oral tissues and a quantitative analysis by flow cytometry of TfR in a cancer cell line after an anticancer drug treatment. TfR was found in the parabasal and basal layers of the normal epithelium, but rarely in benign tumors. Generally, in the malignant tumors, the poor prognostic cases showed strong staining regardless of the differentiation of the tumor. In the flow cytometric analysis, the amount of TfR decreased according to the reduction of the proliferative ability of cancer cells. These results suggest that TfR expression may be useful as a prognostic marker.

Iron is essential for the development of human cells. The transferrin receptor (TfR) plays an important role in iron metabolism, cell proliferation, and malignant transformation of the cells. This receptor is a transmembrane glycoprotein composed of two disulfide-bonded subunits1'5. In vitro studies in nor-

mal and malignant tumor cells have shown that the presence of TfR might indicate the cell-proliferation potential; thus, TfR is a potential marker for the identification of the dividing cells 7. We did an immunohistochemical study of the expression of TfR on oral tissues and a quantitative determi-

Key words: transferrin receptor; oral tumor; immunohistochemical analysis; flow cytometric analysis. Accepted for publication 1 January 1994

nation of TfR content in a cancer cell line (Hela cells).

Material and methods Tissue samples used for the immunohistochemical study were obtained from patients with oral tumor at biopsy or operation. Six-

Fig. 1. Immunohistochemical staining of TfR in normal gingiva. Posi-

Fig. 2. Squamous cell carcinoma of mandible with poor prognosis.

rive reaction was found in cells of parabasal and basal layers (× 130).

Strong staining of TfR may be seen (×210).

Transferrin receptor in oral tumors

Fig. 3. S q u a m o u s cell carcinoma of tongue with good prognosis. Clear reaction of T f R is shown on cell surface (arrowheads) (×210).

teen cases were epithelial malignant tumors (15 s q u a m o u s cell carcinomas and one mucoepidermoid tumor), eight were nonepithelial malignant tumors (two malignant melanomas, two malignant fibrous histiocytomas, two osteosarcomas, one fibrosarcoma, and one malignant lymphoma), and five were benign tumors (neurilemmoma, xanthoma, ne-

431

Fig. 4. TfR expression in malignant m e l a n o m a of mandible, showing strong reaction of TfR (arrowheads) (x400).

urofibroma, ameloblastoma, and pleomorphic adenoma). The tissue was fixed with periodate-lysineparaformaldehyde (PLP). Sections were cut from the frozen tissue in a cryostat at 6/~m and stained with an avidin-biotin-peroxydase complex (ABC). In the flow cytometric analysis, an anti-

cancer drug, cis-platinum diamine dichloride (CDDP), was used at the concentration of 0.1-10.0 /~g/ml. We used an indirect fluorescence antibody method in free cell suspension with FITC-labeled monoclonal antibody against TfR, and we used propidium iodide (PI) to exclude the dead cells. The fluorescence intensity on the axial mode of

Table 1. Immunohistochemical reaction of TfR No.

Sex

Age (yr)

Site

Diagnosis

Epithelial malignant tumors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

M M F F M M M F M F M M M M M F

45 38 32 55 74 45 50 88 53 75 41 54 58 66 43 35

Mandible Tongue Maxilla Tongue Mandible Tongue Tongue Mandible Floor of m o u t h Cheek Tongue Tongue Tongue Floor of m o u t h Tongue Palate

Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell carcmoma Squamous cell carcinoma Mucoepidermoid tumor

Nonepithelial malignant tumors

1 2 3 4 5 6 7 8

F F M M F M F F

30 35 30 56 64 77 0 65

Maxilla Mandible Maxilla Palate Mandible Mandible Mandible Palate

Malignant m e l a n o m a Malignant m e l a n o m a Malignant fibrous histiocytoma Malignant fibrous histiocytoma Osteosarcoma Osteosarcoma Fibrosarcoma Malignant l y m p h o m a

1 2 3 4 5

M F F M M

59 61 19 51 48

Neck Mandible Mandible Tongue Palate

Neurilemmoma Xanthoma Ameloblastoma Neurofibroma Pleomorphic adenoma

Benign tumors

- negative; + positive; + + strongly positive; ~ fatal case.

Degree of Recurrence Degree of differentiation or metastasis staining Poor Well Well Well Well Well Well Well Poor Well Well Well Well Well Well

+~ +~ +t +t +~ +~ + + -

++ ++ + ++ + + + + ++ + + + + + + +

+t

++

+?

+

-

+

-

+

-

+ +

-

+

432

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~ontrQI l . . . . , . . . . . . . . . \~,+,

Control

/I;;7l," II\

I00

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ItR

i1'71'1

I00 Tf£

200

;00 TtR

200

lO0

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100 TrR

200

100 TfR

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Fig. 5. Expression of TfR by flow cytometry after addition of CDDR

cell count was determined by FACStar® as the amount of TfR in the test material. Results Immunohistochemically, in the normal epithelium, the parabasal and basal layers strongly reacted with TfR (Fig. 1). In benign tumors, no staining was observed except in ameloblastoma. Ameloblastoma, which has a high potential for transformation into a malignant tumor, showed diffuse and slight staining. However, this reaction was much weaker than that in the parabasal and basal layers of the normal epithelium. Thus, the reaction in all of the benign tumors examined was classified as negative. All the samples of malignant tumor had staining comparable with or stronger than that in the parabasal and basal layers of the normal epithelium. In the poorly differentiated squamous cell carcinomas, positive staining appeared diffusely on the cell surface, and the degree of the staining was remarkable (Fig. 2). In the well-differentiated squamous cell carcinomas, for which clinical prognosis was poor, staining was detected, as well as in the poorly differentiated squamous cell carcinomas. The other tumors which had good prognosis showed only a weak reaction (Fig. 3). In the nonepithelial malignant tumors, all the samples showed strong evidence of staining for TfR. The stained tumor cells were distributed diffusely, unlike the pattern shown in the epithelial malignant tumors. In tumors for which

the clinical prognosis was poor, the evidence of staining was stronger than in those which had good prognosis. These findings were the same as in the epithelial malignant tumor (Fig. 4). Generally, in the malignant tumors, those with poor prognosis showed diffuse staining regardless of the differentiation of the tumor (Table 1). The flow cytometric study showed that the fluorescence intensity of the cancer cells decreased according to the effect of an anticancer drug, CDDP (Fig. 5). The fluorescence intensity of the cells in the CDDP 0.5-/xg/ml group was not different from that in the control group. The intensity in the 1.0-/xg/ ml, 24-h group was almost the same as that in the control group, but in the 1.0/xg/ml, 48-h group, a decrease of 18% and in the 1.0-/xg/ml, 72-h group a decrease by 10% were observed. The intensity in the 2.0-/xg/ml, 24-h group decreased by 10% and in the 2.0-/xg/ml, 48- and 72-h groups, it decreased more radically, but some dead cells were recognized. Discussion TfR is commonly thought to be expressed on all proliferating cells 6. Immunohistochemically, TfR expression was examined in human breast tissue and human cervical epithelium2,3,8. These results indicated that the malignant tissues and rapidly proliferating tissues manifested strong staining of TfR. In the present study, we examined the evolution of TfR expression in oral ma-

lignant tumors. The positive reaction was seen in all malignant tumors. Generally, it was expressed on the surface of the cell in the epithelial malignant tumors which had a good prognosis. Among the nonepithelial malignant tumors and squamous cell carcinomas, those with poor prognosis, regardless of the degree of the differentiation, showed strong staining in all parts of each cell of the tumor. MAKINO et al. 4 found n o relationship between the expression of TfR and the degree of differentiation of the cells. Our results support ,their conclusion. WILLINGHAM • PASTAN reported 9 that T f R t s concentrated in the clathrincoated pits of the cell surface which transport transferrin into the cell. Our results suggest that, in tumors which have a poor prognosis, many coated pits may appear on the cell surface. At the moment, we are conducting an immunoelectron-microscopic study in oral tumors to determine the localization of the receptor. Our flow cytometric study revealed that the amount of TfR decreased according to the reduction of the proliferation of the tumor cells; thus, it might be possible to indicate objectively the degree of the malignancy in a tumor. From now on, quantitative analysis of TfR in clinical cases will be helpful in establishing the prognosis. We conclude from this study that a large amount of TfR exists in the proliferating tumor cell, and that the investigation of TfR expression seems to be useful in predicting the clinical course of the case. References 1. AISANP, BROWNEB. Structure and function of transferrin. Prog Hematol 1982: 9:25 6.

2. FAULKWR Hsr BL, STEVENSPJ. Transferrin and trans~errin receptor in carcinoma of the breast. Lancet 1980: 23: 39~2. 3. LLOYDJM, DOWDTO, DRIVERM, TEED. Demonstration of an epitope of the transferrin receptor in human cervical epithelium - a potentially useful cell marker. J Clin Pathol 1984: 37: 131-5. 4. MAKINOY, TE~q, KANAMURAI, TAKEDA S, KONNOK. Changes of the number of transferrin receptors during cell differentiation and proliferation. Amsterdam: Elsevier, 1988. 5. SELIGMAN PA, SCHLEICHER RB, ALEN RB. Isolation and characterization of the transferrin receptor from human placenta. J Biol Chem 1979: 254: 9943-6.

Transferrin receptor in oral tumors 6. TROWBRIDGE IS, NEWMAN R A . Monoclonal antibodies to transferrin receptor. In: GREAVES MF, ed.: Antibodies to receptors: probes for receptor structure and function. London: C h a p m a n and Hall, t985:235 62. 7. URUSHIZAKI g. Studies on transferrin receptors of h u m a n cultured cell lines. Sapporo Med J 1983: 52: 269-83.

8. WALKER R A , DAY SJ. Transferrin receptor expression in non-malignant and malignant h u m a n breast tissue. J Pathol 1986: 148: 2 1 7 ~ 4 . 9. WILLINGHAM M e , PASTAN I. Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in h u m a n KB cells. J Histochem Cytochem 1985: 33: 59-64.

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Address:

Dr Takao Miyamoto First Department of Oral and MaxilloJacial Surgery Faculty of Dentistry Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku Tokyo 113 Japan