7 Ectopic hormone production

7 Ectopic hormone production A . J. L. C L A R K

Hormone production by a non-endocrine tumour, so-called ectopic hormone production, is a not uncommon phenomenon, although only occasionally is the nature and extent of hormone production such as to cause a clinically apparent syndrome. Apart from these clinically significant aspects, for example as a source of tumour markers, the phenomenon is of great interest from the point of view of gene regulation and turnout biology. The rapidly advancing field of molecular biology has provided several techniques over the past decade for understanding and assessing gene regulation. Until recently these techniques have been applied largelf to the understanding of biological, rather than pathological, questions. Ectopic hormone production therefore provides a tantalizing area of research in which these new techniques are just beginning to be applied to clinical problems. Under these circumstances, unfortunately, complete and clear data are patchy, which makes it difficult to draw common threads from the various pieces of work with each of the principal ectopic hormones. Most articles on this subject begin with a review of the existing theories that have been put forward to explain the phenomenon. Here, I shall refer to them only briefly, not to belittle the excellent scientific work that underlies them, but in order to take a 'fresh' view based on the accumulating evidence provided by the molecular approach. The reader requiring a more detailed assessment of the clinical, pathological and biochemical characteristics of ectopic hormone secretion, or the proposed mechanisms by which these phenomena arise, is referred to several good reviews on the subject (Rees, 1975; Baylin and Mendelsohn, 1980; Orth, 1987). The various hypotheses can be grouped into those that claim that a fundamental change has occurred in one or more genes, such that they are expressed in situations that never would have been possible at any stage of development or, alternatively, theories that claim that the cell from which the tumour originated had the capability of expressing that gene either currently, or at some stage of development. 1. 2.

Random de-repression: suggesting that consequent upon the catastrophe of neoplastic transformation individual genes, or 'integrator units' for groups of genes, become switched on or off (Gellhorn, 1963). The A P U D concept elegantly demonstrated the development of 'endocrine' cells with amine precursor uptake and decarboxylase (APUD) characteristics from the neural crest, and the existence within normal

Bailli~re's Clinical Endocrinology and Metabolism--Vol. 2, No. 4, November 1988

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A . J . L . CLARK non-endocrine tissue of A P U D cells. Ectopic hormone producing tumours, it was suggested, developed from these scattered cells (Pearse, 1980). The neuroendocrine cell hypothesis: essentially a development from the A P U D concept describing the widespread nature of 'endocrine' cells which could give rise to hormone secreting tumours. Not all such cells need be of neural crest origin, or have characteristic A P U D staining properties. De-differentiation and dys-differentiation: such theories accept the widespread nature of neuroendocrine type cells, which, it is proposed, were hormonally pluripotential at an early stage of development. Normal development shuts off much of this endocrine activity but neoplastic transformation induces de-differentiation or dys-differentiation, such that some of their pluripotential characteristics are regained (Baylin and Mendelsohn, 1980).

These last three theories can in some respects be grouped together, in that they all accept the diffuse nature of endocrine tissue, whether it be currently functional or not. The first and last theories would require that a fairly fundamental change in gene regulation takes place. If this were the case, current molecular biological tools should be able to detect such changes. Most peptide hormones have at some time or another been described as being produced by most histological types of tumour. However,the vast majority of instances of ectopic hormone production involve a relatively small number of hormones produced by a relatively small number of turnout types. These are summarized in Table 1. Table 1. The commonest tumour sources for some of the most commonlyencountered ectopic hormones. Hormone/gene

Tumour types

ACTH/ACTH precursors

Small cell carcinoma of the lung Carcinoid tumours Medullary carcinoma of the thyroid Small cell carcinoma of the lung Small cell carcinoma of the lung Squamous cell carcinoma of the lung Renal carcinoma Small cell carcinoma of the lung Other lung tumours Germ cell tumours Gastric, hepatic, pancreatic and urogenital tract tumours

Vasopressin Gastrin-releasing peptide Parathyroid hormone related peptide Calcitonin gene related peptide [3-Chorionicgonadotrophin

TISSUE SPECIFIC GENE EXPRESSION All nucleated cells contain the same genetic information. In any one cell only a tiny fraction of that information will be expressed, but that infor-

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mation determines what that cell becomes. At the present time we have only a partial understanding of the way in which this regulation is organized, and this is confined to perhaps a score of genes. These findings have been excellently reviewed elsewhere (Dynan and Tjian, 1985; McKnight and Tjian, 1986; Maniatis et al, 1987) and will be discussed more briefly here. A number of elements seem common to most genes. Promoters

All actively transcribed genes require a promoter which is located relatively close to the transcription initiation site (usually about 30 bp upstream). Most often this is a short AT-rich region, usually with a sequence similar to TATA, after which this region is named. A specific protein, the TATA box binding protein, binds to this sequence and seems to act to position R N A polymerase on the gene in the correct position to start transcription (Breathnach and Chambon, 1981). Some genes lack a TATA box, and yet are efficiently transcribed. In many such cases an alternative GC-rich element appears to fulfil the same function. These CG box promoters bind an alternative protein known as Spl (Gidoni et al, 1984; Briggs et al, 1986; Kadonaga et al, 1986). They seem to appear frequently in genes that are widely expressed, often at low levels, so-called 'housekeeping genes'. C AA T box

Many genes containing a TATA box also contain a sequence similar to CAAT, usually located about 80 bp upstream of the transcriptional start site (Benoist et al, 1980; Efstradiadis et al, 1980). The role of this region is less clear cut but it seems to improve the efficiency of transcription when it is present. It has recently become apparent that a number of CAAT box binding proteins exist (Raymondjean et al, 1988; Santoro et al, 1988) but their relative functions are not yet clear. Enhancers

These are perhaps the most fascinating promoter elements of all. They are D N A sequences that bind specific proteins and consequently enhance the rate of gene transcription (Khoury and Gruss, 1983; Serfling et al, 1985; Maniatis et al, 1987). They may be located close to, or at a great distance from the gene they regulate. They may lie downstream or upstream of the transcriptional start site and thus may be found either downstream of the entire gene or in an intron within the gene. In the experimental situation they may be excised and reversed, or placed on the opposite D N A strand, and they will still work efficiently. The mechanism by which enhancers work is not clear. There is evidence that having bound a specific D N A binding protein, this protein interacts with the transcriptional complex (the CAAT box and TATA box). This

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probably involves a looping of DNA, which has been captured on the electron micrograph (Ptashne, 1986). Enhancer binding proteins include many factors that confer responses from extracellular or intracellular signals. These include the receptors for all the steroid hormones and vitamin D (Yamamoto, 1985), the receptor for triiodothyronine (T3) (Weinberger et al, 1986) and several proteins that confer cAMP and C kinase-mediated responses (Montiminy and Bilezikjian, 1987; Imagawa et al, 1987; Lee et al, 1987). In these examples, it seems that the binding protein is inactive until bound by hormone or modified by receipt of the intracellular signal, e.g. by phosphorylation. The activated protein acquires high DNA binding affinity and recognizes and binds to the enhancer element. Specific DNA sequences have been identified for many of these enhancer elements. Enhancers may also confer tissue specificity of gene expression. In these cases it appears that a certain tissue contains the enhancer binding protein, which then directs that one or more genes specific to that tissue are expressed. A number of examples of such tissue-specific enhancers have been described. Of particular interest to endocrinologists is the tissuespecific regulation of the growth hormone and prolactin genes, which has recently been the focus of some attention (Bodner and Karin, 1987; Nelson et al, 1988). One gene may have several enhancers, which may confer a number of influences on gene expression. Although poorly understood at present, it seems that positive and negative co-operativity between enhancers may exist, permitting the heterogeneity and complexity of gene expression typical of many tissues.

Inhibitory regions It is no surprise that 'negative enhancers' are sometimes found. Evidence supporting this class of DNA elements came initially from cell fusion experiments in which a cell expressing a specific gene was fused with a cell that did not express that gene, with the consequent loss of expression of that gene. This is most readily explained by the proposition that the non-expressing cell type contained a DNA binding factor that recognized a specific element in the gene in question. Subsequently, specific examples of such DNA elements have been found, although this class of regulatory region does not seem to be as prevalent as positive enhancement. In a number of circumstances, hormone-responsive enhancers may confer a negative response. The exact mode of action in these circumstances is not clear. In the case of the rat prolactin gene it seems that the activated oestrogen receptor interacts with a protein known as Pit 1 bound to DNA, and confers its inhibitory signal (Adler et al, 1988). In the case of the rat pro-opiomelanocortin gene, it has been proposed that the activated glucocorticoid receptor binds to DNA in the region of the CAAT box and consequently may prevent access of the CAAT box binding protein to its binding site (Drouin et al, 1987).

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DNA methylation

Methylation of DNA plays an uncertain role in the regulation of gene expression (Cedar, 1988). The occurrence of methylation in a particular gene is usually detectable by assessment of the digestion pattern created by a number of restriction enzymes which cut only at unmethylated sites. Evidence generated in this way suggests that, in general, highly methylated promoter regions of many genes are associated with non-expression of that gene in that particular tissue. When the dinucleotide CG is methylated on the cytosine residue, methylcytosine so formed can deaminate to form thymine. This mutation is poorly recognized by repair mechanisms, accounting for the low frequency of CG in the genome (Bird, 1986). It is therefore argued that genes containing a high frequency of CG residues are likely to be genes that are generally undermethylated and thus widely expressed. There is significant support for this hypothesis when a large number of genes are assessed, and it becomes clear that housekeeping genes contain multiple 'Hpa II tiny fragment islands' or HTF islands. (Hpa II is a restriction enzyme that cuts at the DNA sequence CCGG, and thus a frequent cutter in a CG-rich DNA sequence.) When mapped out, HTF islands are found most frequently around the promoter regions of widelyexpressed genes, lending support to the idea that methylation has something to do with gene regulation. The minilocus

A further, higher order of control element has recently been identified in the [3-globin gene (Grosveld et al, 1987). These are DNA elements that seem to lie at either end of the [3-globin gene cluster and which have been identified by their sensitivity to DNAse 1 digestion. Factors, possibly topoisomerases, interact with these elements allowing other regulatory elements to gain access to the genome and, as a result, to regulate gene expression with greater precision. In this section I have tried to summarize as briefly as possible the principal factors that are believed to govern gene expression. Many of these factors will be exemplified in the next section.

PEPTIDE HORMONE GENE EXPRESSION IN TUMOURS Pro-opiomelanocortin

Pro-opiomelanocortin (POMC), the 31 kDa peptide precursor of adrenocorticotrophic hormone (ACTH), ~-melanocyte stimulating hormone and t3-endorphin, is the product of a single gene in all species studied (except the mouse which has a single additional pseudogene). The human POMC gene contains three exons interrupted by introns of 3.7 kb and 2.8 kb. The first exon contains the leader sequence, the second exon contains the signal

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peptide sequence and part of the N-terminal portion of POMC, and the third (longest) exon contains the remainder of the coding region (Figure 1). The pituitary is the principal source of POMC-derived peptides but many other tissues are known to produce the peptide, as determined by immunoassay and immunocytochemistry (Krieger, 1983). POMC gene transcripts

I] PROMOTER m

1 --~

I-T-I11121 3

2---~ --

3~1,11121

bAAA

1200b

mRNA

~.-AAA

800b

mRNA

3

1400b

mRNA

~-AAA

Figure 1. Structure of the human pro-opiomelanocortin gene, illustrating the relationships of the three exons (numbered in rectangles), and the three putative promoters (numbered in circles). Promoter 1 is that active in pituitary and most ectopic ACTH producing turnouts, resulting in an RNA transcript as shown. Promoter 3 is responsible for the 5' extension to the RNA transcript (*) found in some ectopic ACTH tumours. Promoter 2 is responsible for the short RNA transcript comprised of all but the 5' end of exon 3, which produces a non-secretable product.

have also been demonstrated in many sites, including the testis and epididymis (Pintar et al, 1984), ovary and placenta (Chen et al, 1986), lung, thymus, adrenal medulla and duodenum (Jingami et al, 1984), lymphocytes (Oates et al, 1988; Buzetti et al, 1989) and colon, liver, thyroid and kidney (DeBold et al, 1988a). The size of POMC m R N A produced in the pituitary is estimated from northern blots to be about 1100-1200 bases in length, as would be predicted if transcription was initiated at the start of exon 1 and ended at the poly (A) addition site of exon 3, with all R N A splicing being performed as suggested in Figure 1. However, the POMC mRNA produced by normal non-pituitary tissues is approximately 800 bases in length. Lacaze-Masmonteil et al (1987) have demonstrated in man, Jingami et al (1984) in cattle, and Jeannotte et al (1987a) in rat (where similar R N A size variations are found), that these shorter transcripts initiate at a number of sites at the beginning of exon 3. This R N A species lacks those regions encoding the leader sequence and signal peptide. Thus, if the message were translated the resultant peptide would not be expected to have gained access to the endoplasmic reticulum, and thus the Golgi apparatus and secretory vesicle, and therefore should not be secretable. In vitro we have found that this short transcript is translatable, producing two peptides of 27.5 and 24 kDa. Evidence that this occurs in vivo comes from the data reporting the detection of POMC peptides in normal

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non-pituitary tissues (see above) but it is argued that a small, efficiently translated quantity of full length mRNA might co-exist with the shorter message and that this is responsible for the peptide. Transfection of gene fusions and generation of a number of permanent cell lines has allowed us to show that this short mRNA is translated but does not produce a secretable peptide (Clark et al, unpublished data). It has been proposed that this short POMC transcript is produced by a CG box type promoter found at the 3' end of intron 2 (promoter 2 in Figure 1). At the present time we have been unable to demonstrate any promoter function in a 1.2 kb fragment containing this putative promoter, by means of gene fusion/transfection studies, but this may reflect its low level of activity. The full length (pituitary) mRNA appears to be generated by a TATA box type promoter located upstream of exon 1 (promoter 1 in Figure 1). In man, there is less data on other regulatory regions in this vicinity, although it has been demonstrated that this is a functional promoter in vitro (Notake et al, 1983) and in vivo (Mishina et al, 1982). Moreover, the DNA segment spanning 670 bp upstream of the mRNA cap site has been shown to confer glucocorticoid inhibition of transcription (Israel and Cohen, 1985). Considerably more detailed analysis of the rat POMC promoter has been performed. Two groups have shown that two regions confer tissue-specific enhancement of gene expression (Jeannotte et al, 1987b; Roberts et al, 1988). Several regions that bind glucocorticoid receptors in vitro have been identified. In vivo, it appears that only one of these, located at - 6 3 bp relative to the start of transcription, is required (Drouin et al, 1987). This negative glucocorticoid response element overlies a putative CAAT box and possibly may act by steric hinderance of the positively-acting CAAT box binding protein. The current understanding of POMC gene regulation has recently been reviewed in some detail (Lundblad and Roberts, 1988). Ectopic ACTH producing tumours also express the POMC gene but exhibit a variety of mRNA species, as illustrated in Figure 2. Of 17 such tumours from patients with documented ACTH and cortisol excess reported in the literature, the majority produced a POMC transcript corresponding in size to that in the pituitary (lane 1, Figure 2). Several of these tumours also produced a larger RNA species of 1400-1500 bases (Tsukada et al, 1981; DeBold et al, 1983; deKeyzer et al, 1985; Clark et al, 1989) exemplified by the upper band in lane 5, Figure 2. Since this longer mRNA is usually found as a minor species compared to the 1200 base message, it has been difficult to obtain conclusive evidence as to its origin. However primer extension and $1 nuclease protection analysis have suggested it derives from a transcript initiating at about - 360 bp relative to the normal initiation site (Clark et al, 1987). A potential promoter sequence (promoter 3 in Figure 1) exists upstream of this region having a TATTTA sequence (resembling the TATA box) at -392, and a CAAT sequence (a typical CAAT box) at -432. However, this promoter has not yet been demonstrated to be functional in any system. Nevertheless, the majority of transcription appears to initiate from the pituitary promoter, and it is obviously activity of this promoter that is giving rise to the clinically significant secretion of ACTH and other POMC-derived

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Figure 2. Northern blot of human RNA probed with a bovine POMC cDNA probe. Lane 1, normal pituitary; lane 2, an ACTH-secreting lung carcinoid; lane 3, a hepatic metastasis from a pancreatic carcinoid, not shown to be producing ACTH; lane 4, a pituitary corticotrophic tumour; lane 5, an ACTH-secreting lung carcinoid exhibiting the large mRNA transcript (upper arrow). The slight extra length of the RNA species in lane 2 is the result of a longer poly(A) tail than in normal pituitary.

peptides. In one cell line that we have examined, (White et al, 1989) and in one solid tumour (Clark et al, 1989) a long m R N A appears to exist in isolation. A transcript initiating at - 360 would still use the same A T G start codon, and thus no new or additional amino acid sequence would be found in a peptide derived from this message. A further source of POMC m R N A size heterogeneity is illustrated in Figure 2, lane 2. RNAse H analysis of the poly(A) tail length suggests that some ectopic ACTH-producing tumours contain a POMC message with a longer than usual poly(A) tail (Clark et al, 1989). The significance of this is unknown, although a long-tailed POMC message has also been described in the rat hypothalamus (Jeannotte et al, 1987a). An analysis of the POMC gene in human ectopic ACTH-producing turnouts suggests there is no obvious amplification or rearrangement of the gene. There are, however, DNA methylation appearances characteristic of an expressed gene (Lavender et al, 1989). There are a number of reports in the literature of human turnouts not associated with ectopic ACTH secretion containing small quantities of the peptide (Ratcliffe et al, 1972; Gerwitz and Yalow, 1974; Bloomfield et al,

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1977). Recently, we have examined the RNA from several lung tumours (mainly of squamous cell type) and found detectable quantities of an 800 base POMC mRNA. Similar findings are reported by DeBold et al (1988a,b) in several phaeochromocytomas and other tumours. Peptides produced by such a message should not be secretable according to our data, and thus these patients should not develop the ectopic ACTH syndrome. DeBold et al (1988b) also described the co-existence of a long (1400-1500 base) mRNA with these short transcripts and they demonstrated that they probably initiated at, or near, the - 3 6 0 bp transcription initiation site occasionally seen in ACTH-producing turnouts. Therefore, it appears that not only do a significant number of cells in many normal tissues express the POMC gene but many tumours do so as well. The event that determines whether a tumour becomes an ectopic ACTHproducing tumour may be the switching of activity from the putative downstream CG box promoter to the more conventional pituitary promoter. Thus, in summary, the evidence provided from ectopic POMC production is that the gene is very widely expressed in normal adult tissues, but expressed in such a form that peptide is not secreted. A promoter switch, for reasons that can be speculated upon later, may be the trigger that gives rise to this phenomenon.

Vasopressin Ectopic expression of vasopressin (VP) has been shown to occur from a number of tumour types, including small cell carcinoma of the lung (SCLC). Inappropriate production of the peptide, producing water intoxication, may occur in isolation or in addition to other ectopic hormone syndromes. Vasopressin, like POMC, has been shown to occur in many normal tissues other than the hypothalamus, including the adrenal (Nussey et al, 1984), testis (Kasson et al, 1985), and ovary (Lira et al, 1984). Detection of the VP mRNA in normal tissues has been studied in less detail but Fuller et al (1985) clearly demonstrated VP expression in the rat ovary using northern blot hybridization techniques. Vasopressin is derived from a precursor, often referred to as vasopressin/ neurophysin II (VP/NP2), which is positioned in the genome in close association with the oxytocin/neurophysin I (OT/NP1) gene. The two, very similar sequences probably arose by a gene reduplication at some point in evolution and it is interesting to note that the direction of transcription of the two genes is opposed (Figure 3) (Sausville et al, 1985). Although the coding regions of these genes are very similar, the presumed promoter regions show very little homology. (These DNA sequences have not yet been demonstrated to have promoter function.) A CAAT box and a TATA box is found in each at the appropriate positions relative to the transcriptional start site, and both promoters are fairly GC rich (69% for VP/NP2 and 60% for OT/NP1 in the 175 bp most proximal to the transcriptional start site). However, the frequency of the CG dinucleotide is only moderate in both genes. No true Spl binding sites are found in this area.

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Sausville et al (1985) studied a SCLC cell line that produced large quantities of VP and determined that it produced two VP/NP2 mRNA species: a 2.5kb, presumably unprocessed, message and a mature 700 base message--a size predicted from the cloned human genomic sequences. This cell line did not express the OT/NP1 gene as determined by Northern blot analysis.

VP/N P2

OT/N P 1

Figure 3. Intron-exon structure of the vasopressin/neurophysin II (VP/NP2) gene and the closely related oxytocin/neurophysin I (OT/NP1) gene. The arrows indicate the direction of transcription. The genes are separated by a distance of 12 kb.

This specificity is remarkable in view of the proximity of the two genes. Clearly, any enhancer-like effect on VP/NP2 is completely inactive on the OT/NP1 gene. A detailed study of the promoters of these genes would be of great interest.

Gastrin releasing peptide/bombesin Gastrin releasing peptide (GRP) has been shown to be widely distributed in normal tissues, being found in the brain, lung and gastrointestinal tract of man and mammals. The peptide is very frequently produced by SCLC (Moody et al, 1981) and medullary carcinoma of the thyroid (Ghatei et al, 1985), but is not usually associated with any obvious clinical syndrome. The human GRP eDNA was initially cloned from a carcinoid tumour (Spindel et al, 1984) and this was used to clone the human gene (Spindel et al, 1987). The gene contains three exons. Exon l contains the leader sequence, signal sequence and 23 of the 27 amino acids of GRP, while exon 2 contains the remainder of GRP and most of the C-terminal extension peptide. Exon 3 contains the remainder of this extension peptide. The promoter region contains a typical TATA and CAAT box, and it has been suggested that two Spl binding sites are present, although one of these does not conform exactly to the consensus binding sequence (Briggs et al, 1986). This region is relatively GC rich and does contain a high frequency of CG dinucleotide pairs. A putative binding site for the cAMP responsive element (Montminy et al, 1986) is found 175 bp upstream of the transcription initiation site, although the gene is not known to be regulated by this intracellular signal. A GRP-producing SCLC cell line and a lung carcinoid tumour produce a GRP m R N A of similar size to that in brain, stomach, colon and fetal lung (Spindel et al, 1987). This peptide thus represents a further example of a widely expressed gene being transcribed from its usual promoter in tumours.

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Parathyroid hormone related peptide This recently characterized peptide is discussed in greater detail in Chapter 9. Little is known of its normal distribution and function, other than that both the peptide and the m R N A (Mangin et al, 1988) are produced by normal human keratinocytes. Overproduction of the peptide by a variety of tumours, often squamous cell carcinomas (especially of the lung), is one of the major causes of hypercalcaemia of malignancy. The 141 residue peptide and its 36 residue signal sequence are probably the sole products of the gene, although Mangin et al (1988) infer that an additional uncharacterized C-terminal peptide may occasionally be encoded by the mRNA. The gene sequence and organization has not yet been published and thus no information as to the nature of the promoter region is available. The gene appears to exist as a single copy located on chromosome 12 (p11.2-p12.11). As mentioned, the m R N A has been identified in normal keratinocytes as three species of 1.6, 2.1 and 5kb. Similar-sized transcripts have been described in four parathyroid hormone related protein (PTHrP)-expressing renal carcinoma cell lines (Mangin et al, 1988) and in a squamous cell carcinoma of the lung cell line (Suva et al, 1987), which also secretes calcitonin (Ellison et al, 1976; Moseley et al, 1987). Calcitonin/calcitonin gene related peptide Calcitonin (CT), a 32 amino-acid peptide, is normally produced by the C cells of the thyroid and acts to lower calcium. It was shown by Rosenfeld et al (1984) that a second peptide could also be produced by the same gene by alternative splicing (Figure 4). This peptide has been named the CT gene related peptide (CGRP), of which it is the e~ variety; a highly homologous ~-CGRP derived from another gene having been identified subsequently (Steenberg et al, 1985). a-CGRP is expressed more widely than CT, being

CALCITONIN 1000b

CGRP 1100b

Figure 4. Intron-exon structure of the calcitonin/CGRP gene and (below) diagrammatic representation of the alternative splicing that givesrise to the two peptides.

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found in the C cells of the thyroid as well as the central and peripheral nervous systems, especially in relation to blood vessels. This 37 amino-acid peptide is a potent vasodilator, and may act to modulate synaptic transmission. No clinical syndrome resulting from either CT or CGRP excess has been described but production of CT has gained prominence as a tumour marker in the diagnosis and management of medullary carcinoma of the thyroid (MCT). Secretion of CT from this tumour is not regarded as ectopic secretion. Lung tumours, notably small cell lung carcinoma, are the commonest source of ectopic CT and CGRP production. The CT/CGRP gene is located on the short arm of chromosome 11. The gene contains six exons, as illustrated in Figure 4, of which the first three are common to both R N A products. Exon 4 contains the coding region, 3'untranslated region and poly (A) addition signal for CT, while exons 5 and 6 contain the equivalent regions of CGRP. Production of CGRP requires splicing out of the fourth exon during processing of the primary transcript of the gene. Neural tissue which produces CGRP favours this mode of splicing, in contrast to the thyroid C-cell which mainly produces the CT mRNA with a small proportion of CGRP message. Cote and Gagel (1986) demonstrated that in the human TT cell MCT cell line, dexamethasone shifted the splicing preference further in favour of CT. A number of human tumours and human SCLC cell lines have been studied from the point of view of their CT/CGRP m R N A content. Edbrooke et al (1985) studied two SCLC cell lines that were known CT producers and showed the presence of both the 1 kb CT m R N A and the 1.1 kb CGRP mRNA, the former being in significant excess. Nelkin et al (1984) studied ten solid lung tumours (six small cell carcinomas, one large cell carcinoma, two adenocarcinomas and one squamous cell carcinoma) and found that all ten contained the CT mRNA. However, Hoppener et al (1986) also studied a number of tumours not known to produce CT or CGRP. One phaeochromocytoma produced both messages and one adenocarcinoma of the lung contained the CT message but twelve other primary lung tumours, and ten lung tumour metastases were not found to contain either message by northern blot analysis, mRNAs that were detected were of the same size as those found in MCT. Normal lung studied in the same way by this group was also found to be negative for both mRNAs but normal thyroid was shown to contain the CT mRNA.

[3-Chorionic gonadotrophin The chorionic gonadotrophin (CG) glycoprotein hormone comprises an and a [3 subunit encoded by entirely different genes. The o~ subunit is common to all of the glycoprotein hormones (thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone and CG), while the subunit is specific to each. CG is characteristically a placental product but lower levels have been described in a number of normal tissues, including the testis, kidney, liver, lung, stomach and placenta (Braunstein et al, 1975; Yoshimoto et al, 1977, 1979).

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A number of tumour types may produce the [3 chain of CG, with or without the o~ chain. These include germ cell tumours (e.g. of the testis or ovary) and choriocarcinoma. Less well recognized is the frequent production of [3-CG by gastric, hepatic and pancreatic tumours (Baylin and Mendelsohn 1980; Heyderman et al, 1985; Seppala et al, 1986) and by bladder cancer (Dexeus et al, 1986; Iles et al, 1987) and cervical cancer (Patillo et al, 1977). [3-CG is the product of a gene within a gene cluster of six [3-CG-like genes and the [3-LH gene, all located in a region of about 40 kb on chromosome 19 in man (Figure 5). It is seen that in this cluster two pairs of genes are

t-t-_]

,933, 40kb

Figure 5. Map of the human [3-chorionic gonadotrophin/[3-LH gene cluster indicating the relative location of the genes and pseudogenes, and their orientations. [3-CG gene 3 and [~-LH are the only genes which are definitely known to be expressed.~

arranged such that the direction of transcription is opposed. In normal placenta it appears that only one of these genes is definitely expressed (gene 3) as determined by analysis of 15 independently isolated [3-CG clones obtained from a human placental cDNA library (Talmadge et al, 1984). This same group used an alternative approach to determine which genes are expressed and transfected each of the genes into monkey COS cells. It was found that only genes 3 and 5 were capable of being expressed in this system, although this cannot be taken as conclusive evidence that the other genes are unexpressible. A detailed analysis of the promoters of these genes is awaited, although comparison of the available D N A sequence suggests there is very considerable homology between expressed and non-expressed [3-CG genes. There is no published work identifying which of these genes is expressed in tumours, although one might guess that it would be gene 3. It has been shown by Iles et al (1989) that there is an absence of gene rearrangement or amplification in these tumours, as determined by Southern blot analysis. CONCLUSIONS If one is correct in assuming that the mechanisms that produce 'ectopic' hormone secretion are common to the various types of tumours and their products, a number of common threads can now be drawn together. 1.

Tumour-produced hormones are much more widely expressed in normal non-endocrine tissue than are hormones which are rarely expressed. There is little evidence, for example, that growth hormone,

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prolactin, TSH, oxytocin or parathyroid hormone are widely expressed, and there are only rare descriptions of their ectopic production by tumours. . There is no consistent evidence, in those cases that have been examined, of structural alterations of peptide hormone gefies or of their amplification in tumour cells. Some methylation differences may exist in tumours, but this is probably consistent with the fact that the gene is being expressed (Wong et al, 1984; Baylin et al, 1986; Lavender et al, 1989). . Transcription of these genes occurs from the conventional promoter, giving rise to normal-sized transcripts. The possible exception to this, the POMC gene, will be discussed further. The hypothesis that an alternative promoter is active in these tumours is not supported by the data. Thus, normal genes are being transcribed normally in the tumours in question. Since these peptides are seen to be widespread in normal tissue, these observations fit well with the neuroendocrine cell hypothesis of ectopic hormone production, suggesting that the tumour that secretes a peptide is simply a tumour that has arisen from one of the widespread neuroendocrine cells that normally may produce the peptide (or peptides) in question. However, such conclusions ignore the fact that the production and secretion of hormones is a regulated process. Ectopic hormones appear to be produced in an unregulated manner, at least as judged by the clinical effects they can produce. When these peptides are synthesized and secreted from their normal sources, overproduction is prevented by inhibition of a positive stimulatory factor and/or negative feedback by the hormone itself, or some product of its action. In the case of some ectopic hormones it seems reasonable to assume that the production of large quantities of peptides occurs because normal anatomically-located factors are unable to act. For example, a lung tumour producing vasopressin will be unresponsive to neurally-mediated hypothalamic factors such as the signals generated by hypo-osmolality. In other cases, it is conceivable that the peptide-producing tumour cell will lack the cellular receptor for a regulatory molecule. For example, calcitonin inhibition by vitamin D (Cote et al, 1987) may not occur because the tumour cell lacks the vitamin D receptor. In at least one example, the ectopic ACTH syndrome, such explanations for the lack of negative feedback are difficult to believe because the modulator of this feedback is the highly ubiquitous glucocorticoid receptor. An attempt to define a model for ectopic POMC production has resulted in our characterization of a number of human small cell lung cancer cell lines, all of which synthesize and secrete POMC precursors with very small amounts of ACTH (Stewart et al, 1989; White et al, 1989). One of these lines (COR L103) we have studied in detail from the point of view of glucocorticoid feedback. When incubated in the presence of high concentations of dexamethasone (1 txM) for periods of 1-10 days, no change is seen in the accumulation of POMC peptide or the levels of POMC mRNA. Moreover,

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in these same cells no response is seen to glucocorticoid in the level of expression of either the tyrosine amino transferase gene (which should be stimulated by dexamethasone) or the glucocorticoid receptor gene (which should be inhibited by this treatment). This suggests a global failure of glucocorticoid responsiveness in these cells. However, glucocorticoid receptors appear to exist in these cells as judged by competable glucocorticoid binding in both the cytosol and the nucleus, with similar numbers of binding sites and dissociation constants as the apparently functional receptor found in mouse AtT20 cells (Clark et al, unpublished data). This finding is perhaps not as remarkable as it first appears. There are several reports in the literature of steroid unresponsiveness in turnouts in the presence of apparently adequate and normal steroid binding (e.g. Miesfeld et al, 1984; Westphal et al, 1984; Gaubert et al, 1986; Darbre and King, 1987). Returning to the earlier discussion on POMC gene expression in various turnouts, it will be recalled that several non-ACTH secreting tumour types examined express a short POMC mRNA, probably resulting from transcription driven by an intron 2 CG box promoter. The peptide product of this transcript does not appear to be secreted. It seems possible that in turnouts derived from low level POMC expressing cells, gene expression can occur in the presence of functional glucocorticoid receptors by making use of the intron 2 promoter. (As discussed, the evidence suggests that the site of glucocorticoid receptor binding to the POMC gene is in the region of the pituitary promoter, probably close to the putative CAAT box. Thus, a relatively distant promoter will probably be fairly insensitive to its inhibitory action.) If, for some undetermined reason, the turnout cells lose their functional glucocorticoid receptors (although steroid binding may be retained), the pituitary promoter becomes de-repressed, allowing the production of a full length mRNA that produces a secretable peptide. Processing of this peptide within the cell, or after secretion, is well recognized as being variably incomplete (Hale et al, 1986; Crosby et al, 1988) but, even when sufficient ACTH is produced to cause glucocorticoid excess, the tnmour lacks the functional receptors to respond. There is a great deal of further work to be done in the field of ectopic hormone production. The increasingly widespread availability of new techniques of investigation make this an exciting time in this field. It seems highly probable that hormone producing tumours do so because their cells of origin produced hormones. However, it is difficult to conceive the type of experiments that would prove this supposition conclusively. It may be that future trends in cell and molecular biology will provide the techniques that would make such experiments possible. SUMMARY

Current understanding of the phenomenon of ectopic hormone production is largely based on a histopathological and immunocytochemical analysis of

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peptide hormone secreting tumours arising in non-endocrine tissues. Recent advances in the study of gene regulation show that the tissue-specific expression of genes is a highly sophisticated process and is unlikely to be disturbed by a spontaneous event such as point mutation in DNA. Study of several genes for frequently found ectopic hormones, i.e. proopiomelanocortin, vasopressin/neurophysin II, gastrin-releasing peptide, parathyroid hormone-related peptide, calcitonin gene-related peptide and f3-chorionic gonadotropin, suggests they are transcribed as they would be in their natural cell of origin. It is argued therefore that these data are compatible with the concept that the tumour cell of origin was capable of expressing these peptides, if only in a minor or transient manner. In one example, the ectopic ACTH syndrome, it is also necessary to explain the non-suppression of this gene's expression by elevated levels of glucocorticoids. Recent work suggests that this may result from physically present, but biologically inactive glucocorticoid receptors, a phenomenon that has occasionally been noted in hormonally inactive tumour tissue and cell lines.

Acknowledgements I would like to thank many people for their help in preparing this chapter, both by way of discussion and provision of unpublished results. They include Professors Michael Besser and Lesley Rees, Dr Anne White, Dr Rowan DeBold, Paul Lavender and Ray Iles.

REFERENCES Adler S, Waterman ML, He X & Rosenfeld MG (1988) Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor-DNA binding domain. Cell 52: 685-695. Baylin SB & Mendelsohn G (1980) Ectopic (inappropriate) hormone production by tumours: mechanisms involved and the biological and clinical implications. Endocrine Reviews 1: 45-77. Baylin SB, Fearon ER, Vogelstein B e t al (1987) Hypermethylation of the 5' region of the calcitonin gene is a property of human lymphoid and acute myeloid malignancies. Blood 70: 412-417. Benoist C, O'Hare K, Breathnach R & Chambon P (1980) The ovalbumin gene--sequence of putative control regions. Nucleic Acids Research 8: 127-142. Bird AP (1986) CpG-rich islands and the function of DNA methylation. Nature 321: 209-213. Bloomfield GA, Holdaway IM, Corrin B e t al (1977) Lung tumours and ACTH production. Clinical Endocrinology 6" 95-104. Bodner M & Karin M (1987) A pituitary-specific transacting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells. Cell 50: 267-275. Braunstein GD, Rasor J & Wade ME (1975) Presence in normal testes of chorionic gonadotropin-like substance distinct from human luteinizing hormone. New England Journal of Medicine 293: 1339-1343. Breathnach R & Cambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annual Review of Biochemistry 50: 349-383. Briggs MR, Kadonaga JT, Bell SP & Tjian R (1986) Purification and biochemical characterization of the promoter specific transcription factor, Spl. Science 234: 47-52. Buzetti R, McLoughlin-L, Lavender PM, Clark AJL & Rees LH (1989) Expression of pro-opiomelanocortin gene and quantification'of ACTH-Iike immunoreactivity in normal human peripheral mononuclear cells, lymphoid and myeloid malignancies. Journal of Clinical Investigation 83: 733-737.

ECTOPIC HORMONE PRODUCTION

983

Cedar H (1988) DNA methylation and gene activity. Cell 53: 3-4. Chen C-LC, Chang C-C, Krieger DT & Bardin CW (1986) Expression and regulation of pro-opiomelanocortin-like gene in the ovary and placenta: comparison with the testis. Endocrinology 118: 2382-2389. Clark AJL, Lavender PM, Besser GM & Rees LH (1987) Abnormal transcription initiationof the pro-opiomelanocortin gene in human ectopic ACTH producing turnouts. Journal of Endocrinology 115 (supplement): 110. Clark AJL, Lavender PM, Besser GM & Rees LH (1989) Pro-opiomelanocortin mRNA size heterogeneity in ACTH-dependent Cushing's syndrome. Journal of Molecular Endocrinology 2: 3-9. Cote GJ & Gagel RF (1986) Dexamethasone differentially affects the levels of ealcitonin and calcitonin gene-related peptide mRNAs expressed in a human medullary thyroid carcinoma cell line. Journal of Biological Chemistry 261: 15524-15528. Cote GJ, Rogers DG, Huang ESC & Gaget RF (1987) The effect of 1,25-dihydroxyvitaminD3 treatment on calcitonin and calcitonin gene-related peptide mRNA levelsin cultured human thyroid C-cells. Biochemical and Biophysical Research Communications 149: 239-243. Crosby SR, Stewart MF, Ratcliffe JG & White A (1988) Direct measurement of the precursors of adreno-corticotrophin in human plasma by two-site immunoradiometric assay. Journal of Clinical Endocrinology and Metabolism 67: 1272-1277. Darbre PD & King RJB (1987) Progression to steroid insensitivity can occur irrespective of the presence of functional steroid receptors. Cell 51: 521-528. DeBold CR, Schworer ME, Connor TB, Bird RE & Orth DN (1983) Ectopic pro-opiomelanocortin: sequence of cDNA coding for ~/-melanocyte-stimulatinghormone and [3-endorphin. Science 220: 721-723. DeBold CR, Menefee JK, Nicholson WE & Orth DN (1988a) Proopiomelanocortin gene is expressed in many normal human tissues and in tumours not associated with ectopic ACTH syndrome. Molecular Endocrinology 2: 862-870. DeBold CR, Mufson EE, Menefee JK & Orth DN (1988b) Proopiomelanocortin gene expression in a phaeochromocytoma using upstream transcription initiation sites. Biochemical and Biophysical Research Communications 155: 895-900. deKeyzer Y, Bertagna X, Lenne F et al (1985) Altered proopiomelanocortin gene expression in adrenocorticotropin-producing nonpituitaryturnouts. Journal of Clinical Investigation 76: 1892-1898. Dexeus F, Ligothetis C, Hossan E & Samuels ML (1986) Carcinoembryonic antigen and [3-humanchorionie gonadotropin as serum markers for advanced urothelial malignancies. Journal of Urology 136: 403-407. Drouin J, Charron J, Gagner J-P et al (1987) Pro-opiomelanocortin gene: a model for negative regulation of transcription by glucocorticoids. Journal of Cellular Biochemistry 35: 293-304. Dynan WS & Tjian R (1985) Control of eukaryotic messenger RNA synthesis by sequencespecific DNA-binding proteins. Nature 316: 774-778. Edbrooke MR, Parker D, McVey JH et al (1985) Expression of the human calcitonin/CGRP gene in lung and thyroid carcinoma. EMBO Journal 4: 715-721. Efstradiadis A, Posakony JW, Maniatis T et al (1980) The structure and evolution of the human [3-globingene family. Cell 21: 653-668. Ellison ML, Hillyard CJ, Bloomfield GA et al (1976) Ectopic hormone production by bronchial carcinomas in culture. Clinical Endocrinology 5: 397-406. Fuller PJ, Clements JA, Tregear GW et al (1985) Vasopressin-neurophysin II gene expression in the ovary: studies in Sprague-Dawley, Long-Evans and Brattleboro rats. Journal of Endocrinology 105: 317-321. Gaubert C-M, Carriero R & Shyamala G (1986) Relationships between mammary estrogen receptor and estrogenic sensitivity. Molecular properties of cytoplasmic receptor and its binding to deoxyribonucleic acid. Endocrinology 118" 1504-1512. Gellhorn A (1963) The unifying thread. Cancer Research 23: 961-970. Gerwirtz G & Yalow RS (1974) Ectopic ACTH production in carcinoma of the lung. Journal of Clinical Investigation 53: 1022-1023. Ghatei MA, Springall DR, Nicholl CG, Polak JM & Bloom SR (1985) Gastrin releasing peptide-like immunoreactivity in medullary thyroid carcinoma. American Journal of Clinical Pathology 84: 581-586.

984

A . J . L, CLARK

Gidoni D, Dynan WS & Tjian R (1984) Multiple specific contacts between a mammalian transcription factor and its cognate promoters. Nature 312: 409-413. Grosveld F, von Assendelft GB, Greaves DR & Kollias G (1987) Position-independent, high level expression of the human [3-globin gene in transgenic mice. Cell 51: 975-985. Hale AC, Besser GM & Rees LH (1986) Characterization of pro-opiomelanocortin-derived peptides in pituitary and ectopic adrenocorticotrophin-secreting tumours. Journal of Endocrinology 108: 49-56. Heyderman E, Chapman DV, Richardson TC, Calvert 1 & Rosen SW (1985) Human chorionic gonadotropin and human placental lactogen in extragonadal tumours: an immunoperoxidase study of ten non-germ cell neoplasms. Cancer 56: 2674-2682. Hoppener JWM, Steenbergh PH, Moonen PJJ et al (1986) Detection of mRNA encoding calcitonin, calcitonin gene related peptide and proopiomelanocortin in human tumours. Molecular and Cellular Endocrinology 47: 125-130. Iles RK, Oliver RTD, Kitau M, Walker C & Chard T (1987) In vitro secretion of human chorionic gonadotrophin by bladder tumour cells. British Journal of Cancer 55: 623-626. Iles RK, Czepulkowski BH, Young BD & Chard T (1989) Amplification or rearrangement of the [~-human chorionic gonadotrophin-human LH gene cluster is not responsible for the ectopic production of J3-hCG by bladder tumour cells. Journal of Molecular Endocrinology 2: 113-117. Imagawa M, Chiu R & Karin M (1987) Transcription factor AP-2 mediates induction by two different signal transduction pathways: protein kinase C and cAMP. Cell 51: 251-260. Israel A & Cohen SN (1985) Hormonally mediated negative regulation of human proopiomelanocortin gene expression after transfection into mouse L-cells. Molecular and Cellular Biology 5: 2443-2453. Jeannotte L, Burbach JPH & Drouin J (1987a) Unusual proopiomelanocortin ribonucleic acids in extrapituitary tissues: intronless transcripts in testes and long poly(A) tails in hypothalamus. Molecular Endocrinology 1: 749--757. Jeannotte L, Trifiro MA, Plante RK, Chamberland M & Drouin J (1987b) Tissue-specific activity of the pro-opiomelanocortin gene promoter. Molecular and Cellular Biology 7: 4058-4064. Jingami H, Nakanishi S, Imura H & Numa S (1984) Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA. European Journal of Biochemistry 142: 441-447. Kadonaga JT, Jones KA & Tjian R (1986) Promoter-specifc activation of RNA polymerase II transcription by Spl. Trends in Biochemical Sciences 11: 20--23. Kasson BG, Meidan R & Hsueh AJW (1985) Identification and characterization of arginine vasopressin-like substances in the rat testis. Journal of Biological Chemistry 260: 53025307. Khoury G & Gruss P (1983) Enhancer elements. Cell 33: 313-314. Krieger DT (1983) The multiple faces of pro-opiomelanocortin, a prototype precursor molecule. Clinical Research 31: 342-353. Lacaze-Masmonteil T, deKeyzer Y, Luton J-P, Kahn A & Bertagna X (1987) Characterization of proopiomelanocortin transcripts in human nonpituitary tissues. Proceedings of the National Academy of Sciences of the USA 84: 7261-7265. Lavender PM, Clark AJL, Besser GM & Rees LH (1989) Methylation status of the human proopiomelanocortin gene in normal and neoplastic tissues. Journal of Endocrinology 121: 221 (supplement). Lee W, Mitchell P & Tjian R (1987) Purified transcription factor AP-1 interacts with TPAinducible enhancer elements. Cell 49: 741-752. Lim ATW, Lolait SJ, Barlow JW et al (1984) Immunoreactive arginine-vasopressin in Brattleboro rat ovary. Nature 310: 61-64. Lundblad JR & Roberts JL (1988) Regulation of proopiomelanocortin gene expression in the pituitary. Endocrine Reviews 9: 135-158. McKnight S & Tjian R (1986) Transcriptional selectivity of viral genes in mammalian cells. Cell 46: 795-805. Mangin M, Webb AC, Dreyer BE et al (1988) Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumour associated with humoral hypercalcemia of malignancy. Proceedings of the National Academy of Sciences of the USA 85: 597-601. Maniatis T, Goodbourn S & Fischer JA (1987) Regulation of inducible and tissue-specific gene expression. Science 236: 1237-1244.

ECTOPIC HORMONE PRODUCTION

985

Miesfeld R, Okret S, Wikstrom AC et al (1984) Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312: 779-781. Mishina M, Kurosaki T, Yamamoto T et al (1982) DNA sequences required for transcription in vivo of the human corticotrophin-~qipotrophin precursor gene. EMBO Journal 1: 15331538. Montminy MR & Bilezikjian LM (1987) Binding of a nuclear protein to the cyclic AMP responsive element of the somatostatin gene. Nature 328: 175-178. Montminy MR, Sevarino KA, Wagner KA, Mandel G & Goodman RH (1986) Identification of a cyclic AMP responsive element within the rat somatostatin gene. Proceedings of the National Academy of Sciences of the USA 83: 6682-6686. Moody TW, Pert CB, Gazdar AF, Carney DN & Minna JD (1981) High levels of intracellular bombesin characterise human small-cell lung carcinoma. Science 214: 1246-1248. Moseley JM, Kubota M, Diefenbach-Jagger H et al (1987) Parathyroid hormone-related protein purified from a human lung cancer cell line. Proceedings of the National Academy of Sciences of the USA 84: 5048-5052. Nelkin BD, Rosenfeld KI, de Bustros A et al (1984) Structure and expression of a gene encoding human calcitonin and calcitonin gene related peptide. Biochemical and Biophysical Research Communications 123" 648-655. Nelson C, Albert VR, Elsholtz HP, Lu LIW & Rosenfeld MG (1988) Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 239: 1400-1405. Notake M, Kurosaki T, Yamamoto T et al (1983) Sequence requirement for transcription in vitro of the human corticotrophin/[3-1ipotrophin precursor gene. European Journal of Biochemistry 133: 599-605. Nussey SS, Ang VTY, Jenkins JS, Chowdrey HS & Bisset GW (1984) Brattleboro rat adrenal contains arginine-vasopressin. Nature 310: 64-66. Oates EL, Allaway GP, Armstrong GR et al (1988) Human lymphocytes produce proopiomelanocortin gene-related transcripts. Journal of Biological Chemistry 263: 1004110044. Orth DN (1987) Ectopic hormone production. In Felig P, Baxter JD, Broadus AE & Frohman LA (eds) Endocrinology and Metabolism 2nd edn, pp 1692-1735. New York: McGraw Hill. Patillo RA, Hussa RO, Story MT et al (1977) Tumour antigen and human chorionic gonadotropins in CASK1 cells: a new epidermoid cervical cancer cell line. Science 196: 1456-1458. Pearse A G E (1980) The APUD concept and hormone production. Clinics in Endocrinology and Metabolism 9: 211-222. Pintar JE, Schachter B, Herman AB, Durgerian S & Krieger DT (1984) Characterization and localization of pro-opiomelanocortin mRNA in the adult rat testis, Science 225: 632-634. Ptashne M (1986) Gene regulation by proteins acting nearby and at a distance. Nature 322: 697-701. Ratcliffe JG, Knight RA, Besser GM, Landon J & Stansfeld AG (1972) Tumour and plasma ACTH concentrations in patients with and without the ectopic ACTH syndrome. Clinical Endocrinology 1: 27-44. Raymondjean M, Cereghini S & Yaniv M (1988) Several distinct 'CCAAT' box binding proteins coexist in eukaryotic cells, Proceedings of the National Academy of Sciences of the USA 85: 757-761. Rees LH (1975) The biosynthesis of hormones by non-endocrine tumours--a review. Journal of Endocrinology 67: 143-175. Roberts JL, Lundblad JR, Eberwine JH et al (1988) Hormonal regulation of POMC gene expression in pituitary. Annals of the New York Academy of Sciences 512: 275-285. Rosenfeld MG, Amara SG & Evans RE (1984) Alternative RNA processing: determining neuronal phenotype. Science 225: 1315-1320. Santoro C, Mermod N, Andrews PC & Tjian R (1988) A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature 334: 218-224. Sausville E, Carney D & Battey J (1985) The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. Journal of Biological Chemistry 260: 10236-10241. Seppala M, Iino K & Rutanen EV (1986) Placental proteins in oncology. Clinics in Obstetrics and Gynaecology 14: 593-610.

986

A . J . L . CLARK

Serfling E, Jasin M & Schaffner W (1985) Enhancers and eukaryotic gene transcription. Trends in Genetics 1: 224-230. Spindel ER, Chin WW, Price Jet al (1984) Cloning and characterization of cDNAs encoding human gastrin releasing peptide. Proceedings of the National Academy of Sciences of the USA 81: 5699-5703. Spindel ER, Zilderberg MD & Chin WW (1987) Analysis of the gene and multiple messenger ribonucleic acids (mRNAs) encoding human gastrin releasing peptide: alternate RNA splicing occurs in neural and endocrine tissue. Molecular Endocrinology 1: 224-232. Stewart MF, Crosby SR, Gibson S, Twentyman PR & White A (1989) Small cell lung cancer cell lines secrete high levels of ACTH precursor peptides. British Journal of Cancer (in press). Suva LJ, Winslow GA, Wettenhall REH et al (1987) A parathyroid hormone-related protein implicated in malignant hypercalcaemia: cloning and expression. Science 237: 893--896. Talmadge K, Boorstein WR, Vamvakopoulos NC, Gething MJ & Fiddes JC (1984) Only three of the seven human chorionic gonadotrophin 13 subunit genes can be expressed in the placenta. Nucleic Acids Research 12: 8415-8436. Tsukada T, Nakai Y, Jingami H e t al (1981) Identification of the mRNA coding for the ACTH-13-1ipotrophin precursor in a human ectopic ACTH producing tumour. Biochemical and Biophysical Research Communications 98: 535-540. Weinberger C, Thompson CC, Ong ES et al (1986) The c-erb-A gene encodes a thyroid hormone receptor. Nature 324: 641-646. Westphal HM, Mugele K, Beato M & Gehring U (1984) Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies. EMBO Journal 3: 1493-1498. White A, Farrell WE, Crosby SR et al (1989) Pro-opiomelanocortin gene expression and peptide secretion in human small cell lung cancer cell lines. Journal of Molecular Endocrinology (in press). Wong DTW, Hartigan JA & Biswas DK (1984) Mechanism of induction of human chorionic gonadotrophin in lung tumour cells in culture. Journal of Biological Chemistry 259: 10738-10744. Yamamoto KR (1985) Steroid receptor regulated transcription of specific genes and gene networks. Annual Review of Genetics 19: 209-252. Yoshimoto Y, Wolfson AR & Hirose F (1977) Human chorionic gonadotropin-like substance in nonendocrine tissues of normal subjects. Science 197: 575-577. Yoshirnoto Y, Wolfson AR, Hirose F & Odell WD (1979) Human chorionic gonadotropin-like material: presence in normal tissues. American Journal of Obstetrics and Gynecology 134: 729-733.