Expression of the aromatic l-amino acid decarboxylase mRNA in human tumour cell lines of neuroendocrine and neuroectodermal origin

Abstract

Neuroendocrine differentiation of lung tumours is characterised by the expression of several neuroendocrine markers and is confined mostly to specific histological subtypes, i.e. small cell carcinomas and carcinoids. One of the markers seen in neuroendocrine tumours, high activity of the aromatic l-amino acid decarboxylase (AADC), is helpful in distinguishing the classic and variant small cell lung tumour subtypes. Here, we have analysed the expression and quantified the level of mRNA coding for AADC in human tumour cell lines by use of the reverse transcription and polymerase chain reaction (RT–PCR). High amounts of mRNA were detected in classic small cell lung carcinomas and a neuroblastoma cell line. Other cell lines (melanomas, non-small cell lung carcinomas and osteosarcoma) also showed AADC expression, but the levels were 2–3 orders lower. Also, the tissue-specific (neuronal versus liver-specific) mRNA type has been estimated. Small cell lung carcinomas, neuroblastoma and melanoma expressed messenger RNA specific for neuronal tissues. Importantly, the non-small cell lung carcinoma cell lines expressed either liver-specific (non-neuronal) mRNA (cell line A549) or predominantly the neuronal (cell line NCI-H520) AADC message. These data indicate that a range of tumour cell lines transcribe the AADC gene and that two distinct types of AADC mRNA which reflect the embryonal (neuronal or non-neuronal) origin of the tumour may be produced in non-small cell lung cancer cells.

Introduction

Among lung cancers, classic small cell lung carcinomas (SCLC) and a subset of non-small cell lung tumours (NSCLC) express neuroendocrine markers. Previous studies have shown that dopa decarboxylase, chromogranin A (mRNA and immunoreactivity) and synaptophysin in lung tumour cell lines and specimens are markers for neuroendocrine differentiation1, 2, 3. High dopa decarboxylase activities have been found in classic SCLC that distinguished them from variant types[1]. Other proteins, more or less specific for the neuroendocrine phenotype, are often found in lung cancer4, 5. The most common lung tumours with neuroendocrine differentiation are classic SCLC and carcinoids. However, a subset of NSCLC (about 10–15%) also display neuroendocrine features, including aromatic l-amino acid decarboxylase (AADC) activity. It is believed that the neuroendocrine tumours arise from cells dispersed in the bronchial epithelium which are similar to cells distributed in some other organs and sharing neuroendocrine features.

AADC (EC 4.1.1.28, dopa decarboxylase) catalyses the formation of dopamine from l-dopa as a substrate. The enzyme is also able to convert 5-hydroxytryptophan to serotonin, as well as to decarboxylate several other amino acids, such as tyrosine, tryptophan and phenylalanine, although with lower efficiency, to the corresponding amines[6]. AADC is expressed in both the central and the peripheral nervous systems in neurons producing dopamine and serotonin and in the chromaffin cells of the adrenal medulla. The enzyme is also expressed in neuroendocrine cells distributed in several organs such as lung and intestine (amine precursor uptake and decarboxylation, APUD system) and also in the liver and kidney[6].

The human AADC gene is present as a single copy and its coding region is distributed over 15 exons in the genome[7]. The cDNA has been cloned[8]and shown to code for a protein 480 amino acids long. Rat cDNA that codes for a protein product very similar to the human enzyme has also been cloned[9]. It has been demonstrated that the mRNA transcribed in neuronal and neuroendocrine tissues differs from the non-neuronal type (expressed in liver and kidney) by the untranslated 5′-end10, 11, 12. This difference is due to the alternative promoter usage followed by alternative splicing10, 11, 12, 13. In both rat and human genomic DNA, the non-neuronal promoter is located upstream and the neuronal promoter downstream, closer to the second exon which is common to both tissue systems. In rat tissues, the splicing acceptor in neural cells is located five bases downstream from that in the non-neuronal tissue10, 11. In humans, the different non-neuronal or the neuronal first exons are spliced to a common acceptor site[12]. Thus, human AADC mRNAs have an identical second exon in both groups of tissues.

Recently, a hepatocyte nuclear factor-1 (HNF-1) has been demonstrated to bind to a motif in the rat non-neuronal AADC promoter[14]. The core of this sequence is a short A/T-rich stretch located −49 bp to −35 bp relative to the transcriptional start which is completely homologous to a motif found in a human gene. HNF-1 cDNA, when transfected into AADC non-expressing cells, stimulated transcription from the cotransfected promoter/reporter construct[14]. However, the factors participating in transcription of the neuronal type of AADC are unknown.

Here, we present data demonstrating the expression of AADC mRNA in several types of human tumour cells. Using the quantitative reverse transcriptase–polymerase chain reaction (RT–PCR), even very low levels of the messenger RNA were detected in a range of human tumour cell lines. Using this approach, AADC was found in tumour cell types which have been shown previously to be negative when the dopa decarboxylase activity or Northern blot signals were examined. We also show that, while all SCLC cell lines analysed express a neuronal-type message, NSCLC lines may differ and express either non-neuronal or neuronal forms of AADC mRNA.