ORIGINAL ARTICLE

Genetic variants of calcitonin receptor and breast cancer in Japanese

M. Nakamura, X. Jing, Z. Zhang, H. Yasuoka, Y. Nakamura, T. Yokoi, S. Oura,* T. Sakurai* and K. Kakudo

Second Department of Pathology, Wakayama Medical College, Wakayama, Japan 641-0012. *Department of Surgery,Wakayama Medical College Kihoku Hospital, Wakayama, Japan 649-71

S U M M A R Y. Calcitonin (CT) inhibits the growth of human breast cancer cell lines and calcitonin receptor (CTR) isexpressed in breast cancer lines and breast cancer tissue. In this study, the allele frequencies of the CTR gene were com-pared in the peripheral blood and tumour from 46 breast cancer patients with 50 peripheral blood samples from Japanesewomen. The allele frequencies of CTR gene did not differ between them. There was no significant association of CTR allelefrequencies in invasive breast carcinomas. These results indicate that allelic variation of the CTR gene is not a significantrisk factor for the development of breast carcinoma in Japanese women. Further studies are needed to clarify the role of

hers Ltd

The Breast (1999) 8, 104Ð106© 1999 Harcourt Publishers Ltd

CT and CTR in human breast tissue. © 1999 Harcourt Publis

INTRODUCTION

The calcitonin receptor gene (CTR) has been mapped tochromosome 7q21.3.1,2 CTR has seven potential trans-membrane domains, which are expressed in many organs.We have reported two types of CTR cDNA in humans, one(hCTR-1) having an insertion of 16 amino acids in intra-cellular domain 1, and the other (hCTR-2) without such aninsertion.1,3Ð6 The hCTR-1 and hCTR-2 were first isolatedfrom the cDNA library of both the ovarian carcinoma cellline BIN-67 and the mammary carcinoma cell line T47D,respectively.3,6 In addition, there is a single nucleotide dif-ference at position 1377 of human CTR cDNA, coded eitherC or T in the intracellular domain 4. It was found that thevariant with C at this site was more prevalent among theJapanese population.7 However, it is not clear if there is anyfunctional difference between these variants.

Breast carcinoma is a common cancer. Many factors and some genes are related to this disease. Findlay et al.described CTR expression in several cancer cell lines,including lung and the human breast cancer cell lines(T47D, MCF7 and ZR75).8 Ng et al. reported that calcitonin

10

(CT) treatment of T47D and MCF7 cells in culture potentlyreduced the proliferation of these cells, suggesting that CTcan inhibit growth of human breast cancer cell lines.9 CTR

Address correspondence to: M. Nakamura, Second Department ofPathology, Wakayama Medical School, 811-1 Kimiidera, WakayamaCity, Wakayama, 641-0012, Japan. Tel.: 81 734 41 0635; Fax: 00 81 734 46 4825

might also be an important regulator of growth and diffe-rentiation of the normal mammary tissue.

The aims of the present study were to compare the allelefrequencies of these CTR allelic variants between a group ofbreast cancer patients and controls, and among three com-monly recognised different histological types of invasivebreast carcinoma.

MATERIALS AND METHODS

Blood samples and breast carcinoma tissues

Blood samples were collected as controls from 50 Japanesefemale volunteers. Fourteen blood samples and 46 breastcarcinoma tissues were collected from Wakayama MedicalCollege, Kihoku Hospital and Tamaki Clinic. The diagno-sis of breast carcinoma was made based on general rules forclinical and pathological recording of breast cancer (TheJapanese Breast Cancer Society).

PCR using genomic DNA

DNA was isolated from tissues using a QIAamp Tissue kit(QIAGEN Inc.) and from blood samples using Iso Quick kit(Micro Probe) according to the manufacturerÕs protocol.PCR was performed using a set of primers, the sequences

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of which were 5¢-CTCAGTGATCACGATACTGTG-3¢(forward) and 5¢-ATTCAGTGGAACCAGCGTTGG-3¢(reverse).

PCR amplification was carried out in 40 cycles of denaturation for 30 sec at 95¡C, annealing for 30 sec at57¡C with extension for 30 sec at 72¡C.

Digestion of PCR products

PCR products were digested by AluI restriction enzyme(Nippon Gene Co.) under conditions as recommended bythe manufacturer and then electrophoresed in 12% poly-acrylamide gel and stained with ethidium bromide.

Statistical analysis

Comparisons between groups were performed using the c2

test or FisherÕs exact probability test. P values less than 0.05were considered to be statistically significant.

RESULTS

Forty-six breast cancer patients and 50 controls were geno-typed. PCR amplification using DNA prepared from thesesamples yielding a 228 bp product. These PCR products,when digested by AluI, produced restricted fragments in thefollowing sizes: 228, 120 and 108 bp in C/T genotype atposition 1377 of CTR cDNA; 120 and 108 bp in T/T; and228 bp alone in C/C. The distribution of the individualgenotypes is shown in Table 1.

There was no significant difference in the distribution

of the three genotypes between controls and breast cancerpatients (tumour, P = 0.256 > 0.05, leukocyte, P > 0.999> 0.05). In addition, the distribution of these genotypes was

Table 1 Calcitonin receptor genotypes of breast cancers and controls

Population Total no. No. of individuals with genotypes

C/C C/T T/T

Breast cancer, tumour DNA1 46 42 4 0Breast cancer, leucocyte DNA2 14 12 2 0Control3 leucocyte DNA 50 42 7 1

Allele freequencies compared between population 1 and 3, P = 0.256 > 0.05; population 2 and 3, P > 0.999 > 0.05.

Table 2 Calcitonin receptor genotypes of three histological types ofinvasive breast carcinoma

Histological type Total no. No. of individuals with genotypes

C/C C/T T/T

Papillotubular carcinoma 6 6 0 0Solid-tubular carcinoma 20 18 2 0Scirrhous carcinoma 15 14 1 0

P = 0.716 > 0.05.

not associated with histological types of invasive breast carcinoma-papillotubular, solid tubular and scirrhous carcinoma (P = 0.716 > 0.05)(Table 2).

DISCUSSION

It has been reported that the oestrogen receptor relates togrowth and differentiation of mammary tissues, and is pre-sent in significant amounts in the majority of primary breastcarcinomas.10 Furthermore, it has been reported thatBRCA1 gene is important in familial breast cancer.11 Weexpected CTR to be one of the important regulators ofgrowth and differentiation of normal mammary tissuebecause serum CT is elevated during pregnancy and lacta-tion when mammary gland proliferates and demonstrates theterminal function.12,13 There are numerous reports of a relation-ship between polymorphism of receptor gene and diseases,such as, oestrogen receptor polymorphism and osteo-porosis14 or vitamin D receptor polymorphism and osteo-porosis.15 In this study, we analysed the association between specific CTR polymorphisms and breast cancer. However,we failed to find any significant association between them. These results suggest that variants of CTR may not beof a genetic risk factor for breast cancer in the Japanesepopulation.

CT is known to inhibit gastric acid secretion,16 inhibitosteoclastic bone resorption and stimulate urinary calciumexcretion.17 The concentration of immunoreactive CT inhuman milk is between 10 and 40 times higher than theserum level. These data suggest local production of CT or aspecific transport and concentration mechanism within themammary gland.18 There has been no report on the functionof CTR in the mammary tissues; however, mammarytumour cell lines express a large amount of CTR. In situhybridization analysis of breast cancer samples suggestedthat CTR mRNA is expressed specificaly in tumour tissueand not in surrounding stroma.19 Further studies are neededto clarify the function of CT and CTR in mammary tissue.

Acknowledgements

This work was supported in part by Grants-in-Aid (#07807023) forScientific Research (C) from the Ministry of Education, Science, Sportsand Culture, Japan. We gratefully acknowledge Dr Arata Iwasaki, DrLiang Shan, Mr Osamu Nunobiki, Miss Maki Murakami, and MissEmiko Taniguchi for their assistance.

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