JOURNAL OF PATHOLOGY, VOL.

168: 163-1 68 (1992)

RAPID COMMUNICATION

DIFFERENTIAL EXPRESSION OF THE MITOCHONDRIAL GENE CYTOCHROME OXIDASE I1 IN BENIGN AND MALIGNANT BREAST TISSUE MATTHEW G. F. SHARP*, SUSAN M. ADAMS*, ROSEMARY A. WALKER?, WILLIAM J. BRAMMAR* AND JENNIFER M. VARLEY*'?

*ICI/University Joint Laboratory, Department of Biochemistry, and ?Department of Pathology, University oj Leicester, Leicester LEI 7 R H , U.K. Received 26 March I992 Accepted I0 June 1992

SUMMARY Comparative analysis of expression levels of genes in benign and malignant tumours of the breast has been performed. Differential screening of cDNA libraries identified four genes of the mitochondrial genome as being expressed at different levels in the two tissues compared, but further investigations showed that only the gene encoding subunit 2 of cytochrome c oxidase (COII) is expressed at significantly higher levels in carcinomas compared with fibroadenomas. The mitochondrial genes encoding subunits 2 and 4 of NADH dehydrogenase, and subunit 6 of F,,F,ATPase, were not found to be differentially expressed in carcinomas and fibroadenomas. All four genes were expressed in the epithelium of human breast carcinomas, as shown by in situ hybridization. The expression level of the COII gene is also correlated with carcinoma grade. N o gross alterations to the mitochondrial D N A from these tumours could be detected. The possible implications of these results on the behavioural differences between fibroadenomas and carcinomas are discussed. KEY

WORDS-Breast

cancer, fibroadenoma, metastasis, mitochondrial genes

INTRODUCTION

We have used this technique to differentially screen a cDNA library derived from a fibroadenoma of The development of good markers for metastatic the breast, with complementary cDNA probes potential remains a major goal in cancer research' derived from benign and malignant human breast and progress in this area of research is being made.2 tumours. The results show a consistent relative The increased metastatic potential of certain tumour overexpression of a group of genes encoded by cells is associated with an increased expression of the mitochondrial genome in carcinomas. In situ hybridization studies using fixed tissue sections certain cellular oncogene^.^ The technique of differential hybridization allows have shown that it is the epithelial-derived tumour the identification of nucleic acid sequences present cells which are the site of synthesis of the detected at different levels in related cell populations. mRNA mitochondrial mRNA. species present in similar amounts in both cell types METHODS are effectively cancelled out, and those mRNA species present at different levels are selected. Tissue samples, histology, and nucleic acid preparation Addressee for correspondence: M. G. F. Sharp, Centre for The breast tissues used in this study and the Genome Research, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 354. details of the classification have previously been 0022-3417/92/10016346 $08.00 0 1992 by John Wiley & Sons, Ltd.

164

M. G. F. SHARP E T A L .

Table I-Breast

tumours used in this study

Carcinoma

Grade

c10 C298 C300 C301 C307 C312 C317 C328 C336 C410 C446 C666 C670 C712 C714 C718 C719

I(Tub)

I1 111 111

Node status

S-Phase (YOcells)

Menopausal status

-

NK 14.3 14.0 13.1 6.2 21.0 20.0 23.0 17.0 29.2 NK NK NK NK NK NK NK

Post Pre Peri/post Post Pre Peri/post Post Post NK Pre Post Post Post Post Post Pre Post

+ NK

I1 (IL)

+ + + + + + + +

111 (AM) 111 111

-

I1 111 111 111 111 111 111

I1 (IL) I

+ -

T u b = Tubular carcinoma; IL = infiltrating lobular; AM =atypical medullary. All other carcinoma samples are infiltrating ductal. Lymph node status is shown as either positive (+) or negative (-), as measured by histological evidence of tumour spread to axillary lymph nodes. N K = N o t known. C666 was analysed by in situ hybridization and was not included in the RNA slot blot analysis.

de~cribed,~ and DNA and RNA sample preparation was as described The carcinoma samples and some patient characteristics are listed in Table I. One bilateral and 14 unilateral fibroadenomas were taken from patients ranging between 17 and 47 years of age (mean age = 29.53 years). Preparation and screening of the cDNA library Purification of poly(A)+ RNA7 and the synthesis of complementary DNA8 were followed by incorporation into the vector pUC9 via dG:dC homopolymer tailing. The library was constructed in E. coli, JM83RecA according to Hanahan.g A subpopulation of 12 000 recombinant clones (from a total of 5.6 x lo5 recombinants) was screened by colony hybridization" using cDNA probes as d e ~ c r i b e dAfter . ~ three rounds of screening, a final multiple-probe round allowed 13 comparisons. The results of this round facilitated the selection of cDNAs to be investigated further. D N A sequence analysis Sequencing was performed according to the dideoxy method,'' using either the M13 series of

vectors, or double-stranded template DNA l 2 and the modified T7 DNA polymerase (Sequenase, USB). Hybridization analysis All DNA Southern analysis was performed as described p r e v i o u ~ l y , ~using ? ~ 4 p g of DNA per track. The Hybond-N filters (Amersham) were washed in 0.5 x SSC, 0.1 per cent SDS at 65°C. Northern blots were performed as described.I3 The filters were washed to a stringency of 0.2 x SSC, 0.1 per cent SDS at 65"C, unless otherwise indicated. For RNA slot blots, two RNA samples from each tumour were analysed as described p r e v i ~ u s l yThe .~ details of the adjustments made to allow for unequal loading of the RNA and for the non-linear response of the X-ray film have also been described earlier.4 In situ hybridization The in situ hybridization of 35S-labelled cRNA probes to sections of formalin-fixed, paraffinembedded blocks of resected tumour tissue was as described p r e v i ~ u s l y .Autoradiography ~ was for 4-6 weeks in a dry atmosphere at 4°C.

165

COII EXPRESSION IN BREAST CANCER

Table II-Characteristics and identities of cDNAs selected by differential screening of fibroadenoma- and carcinoma-derived probes

500

I

400

-

-3 300

-

Clone

Size of insert (bp)

F455-3 F455-6

940 910

ND2

F455-4

x 195

ND4

W

F455-2 F4.55-7 F4.55- 13

224 556 x 200

COII

5

F455-8 F455-1 1

440

0

Identity c

224

9?

ATPase 6

Out of I3 clones analysed at the sequence level, eight were shown to be derived from transcripts of mitochondria1 DNA. These clones were analysed further.

RESULTS Screening the cDNA library

The fibroadenoma-derived cDNA library was screened repeatedly by differential hybridization to probes derived from carcinomas and benign fibroadenomas of human breast. From the final round, 13 differentially expressed colonies were selected. These cDNAs were considered the most likely to represent genes consistently expressed at different levels in the majority of individual patients. Nucleotide sequence analysis

Nucleotide sequence analysis was performed on all 13cDNAs. This showed that eight of these clones are derived from the mitochondrial genome, with four different coding regions represented (Table 11). The nucleotide sequence of these cDNAs has not been determined over the whole length, but extensive regions of all clones have been analysed and are identical to the previously published mtDNA sequence.14 All these cDNAs were isolated as being expressed more highly in carcinomas than in fibroadenomas, in at least 14 out of 15 possible comparisons.

Ft 200

0

A

1

100

0 ’ Carcinomas

Fibroadenomas

Fig. I-Relative levels of COII gene expression in breast carcinomas and fibroadenomas, assayed by RNA dot blotting. Laser scanning densitometry data were adjusted for loading by reference to results from a control hybridization with cell like mRNA,4 and expression is given in arbitrary intensity units. The expression levels in carcinomas and fibroadenomas are significantly different (Mann-Whitney U-test, Ho:p ,= p 2 ; H,: p , # p 2 ; P

Differential expression of the mitochondrial gene cytochrome oxidase II in benign and malignant breast tissue.

Comparative analysis of expression levels of genes in benign and malignant tumours of the breast has been performed. Differential screening of cDNA li...
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