Analysis of the tumour suppressor genes, FHIT and WT-1, and the tumour rejection genes, BAGE, GAGE-1/2, HAGE, MAGE-1, and MAGE-3, in benign and malignant neoplasms of the salivary glands

Abstract

Aims: Molecular genetic changes involved in tumorigenesis and malignant transformation of human tumours are novel targets of cancer diagnosis and treatment. This study aimed to analyse the expression of putative tumour suppressor genes, FHIT and WT-1, and tumour rejection genes, BAGE, GAGE-1/2, MAGE-1, MAGE-3, and HAGE (which are reported to be important in human cancers), in salivary gland neoplasms.

Methods: Gene expression was analysed by reverse transcription polymerase chain reaction (RT-PCR) in normal salivary gland tissue and 44 benign and malignant salivary gland tumours.

Results: Aberrant FHIT transcripts were found in one of 38 normal salivary glands, three of 28 adenomas, and two of 16 carcinomas. WT-1 mRNA was detectable in two adenomas and five carcinomas. Immunoblotting showed that WT-1 mRNA expression was associated with raised WT-1 protein concentrations. RT-PCR for detection of BAGE, GAGE, and MAGE gene expression was positive in two adenomas and nine carcinomas, but negative in normal salivary gland tissue. HAGE mRNA was found in two normal salivary glands, 11 benign, and eight malignant tumours.

Conclusions: FHIT mRNA splicing does not appear to be involved in the genesis of salivary gland neoplasms. The upregulation of WT-1 mRNA in tumours of epithelial/myoepithelial phenotype may imply a potential role of WT-1 in the genesis and/or cellular differentiation of these salivary gland tumours. The tumour rejection genes were more frequently, but not exclusively, expressed in malignant salivary gland tumours than in benign neoplasms, although none was suitable as a diagnostic marker of malignancy in salivary gland neoplasms.

Figure 1

(A) Agarose gel electrophoresis showing representative polymerase chain reaction (PCR) products of β actin reverse transcription PCR to demonstrate the integrity of the RNA (top panel). The analysis of FHIT (bottom panel) demonstrated in each case the full length product of 533 bp (lane 1) and in six cases various additional aberrant FHIT transcripts (lanes 2–7). The number of the lane corresponds to the histological diagnosis shown in table 2 ▶. (B) WT-1 mRNA was detectable in two benign and five malignant tumours (lanes 1–7). M, molecular weight marker; N, negative control.

Figure 2

Automatic sequencing of two aberrant FHIT transcripts. (A) This splice variant was found in normal salivary gland tissue and lacked exons 5–8. (B) This variant was detected in a pleomorphic adenoma and demonstrated replacement of exons 4–8 by a non-coding sequence of 46 bp identified in intron 5. The insertion shifted the reading frame and introduced a premature stop codon at the beginning of exon 9.

Figure 3

Immunoblot of tissue lysate from adenoid cystic carcinoma and normal surrounding salivary gland tissue. The 52 kDa WT-1 protein is detected by a polyclonal antibody in tumour tissue lysate but not in normal salivary gland tissue lysate. C, Coomassie staining; M, molecular weight marker (Rainbow High, Amersham, Braunschweig, Germany).

Figure 4

Reverse transcription polymerase chain reaction results of the tumour rejection genes BAGE, GAGE-1/2, MAGE 1, MAGE-3, and HAGE showing products of appropriate size for each gene. M, molecular weight marker; N, negative control.