Mokular and ~e~~u~~~E~~~noio~, 71 (1990) I-12 J.%evier Scientific Publishers Ireland, Ltd.

MOLCEL 02280

Review

Ectopic hormone production by small cell undifferentiated

carcinomas

Pamela J. Russell *, Shaun M. O’Mara * and Derek Raghavan Urob@caI Cancer Research Unit, Royal Prince &bred Hospital, Sydney, ~~trali~ Sydney, Australia

Intmduction &topic production of polypeptide hormones constitutes the major b~~he~cal marker of small cell undifferentiated carcinoma (SCUC), which represents approximately 25% of lung cancers (SCUCL), and also occurs rarely in other tissues, including the prostate gland (SCUCP), bladder, stomach, salivary gland, and pancreas ~Sorenson et al., 1981, 1985; Springall et al., 1986; Jelbart et al., 1988a). Recently, it has been shown that hormone secretion, in particular, gastrin-releasing peptide (GRP) produced by SCUC of the lung, may also have a role in autocrine regulation of cancer cell growth (Carney et al., 1983, 1987; Gaudino et al., 1988). Given these findings, this paper aims to review what is known of the occurrence of ectopic hormone production by SCUC and to examine those factors which regulate hormone production in these cancer cells. SCUC are characte~stic~y composed of histologically uniform, small round cells which express neuroendocrine tumour antigens, and in most, but not all cases, produce a variety of peptide hormones, associated morphologically with the presence of neurosecretory granules (Pearse and Pollak, 1971). Despite marked sensiti~ty of the tumours to radiotherapy or cytotoxic chemotherapy, prognosis is poor, with the median survival for patients with clinically non-metastatic tumours being less than 3 years.

Address for wrrespondence: Pamela J. Russell, The Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown NSW 2050, Australia. * Current address: The Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown NSW 2050, Australia. 0303-7207/90/$03.50

and Department

of Surgery, Universiry

of@imy,

Other turnours, termed ‘carcinoid’, also express characteristics of endocrine-paracrine cells (socalled amine-precursor uptake and decarboxylation, or APUD cells), and may form part of a continuum of differentiation between adenocarcinemas and SCUC (Almagro et al., 1985; Jelbart et al., 1988b). &topic hormone pr~uction by SCUC The pr~uction,of ectopic hormones by SCUC has been widely reported, predominantly as case studies, but tumour models, including cell lines in tissue culture and xenografted human tumours, have been more extensively studied. Many reports refer to radio~unoassay of tumour cell homogenates or immunoperoxidase staining of tumour cells, whilst others are based on presumptive ectopic hormone production in tumour-bearing patients following analysis of blood samples. In one study, 83% of SCUCL produced at least one peptide (Yamaguc~ et al., 1985), whereas many tumours, including those of non-pulmonary origin, produced multiple hormones from a range of different precursors (Table 1A and B), with bombesin (BN), pro-opiomelanocor~ (POMC)-derived peptides {ACTH, ~-endows), calcitonin {CT) and somatostatin (SS) being the most common. Cases of SCUC have been described in which no ectopic hormone production has been detected, and conversely, tumour hormone production has not been exclusive to small cell carcinomas; occasionally bronchial adenocarcinomas (Strott et al., 1968; Yamaguchi et al., 1986) have been shown to produce ACTH, somatostatin and calcitonin gene-related peptide (CGRP). Though the range of peptide hormones produced by SCUC is extensive (Table 1A and B),

Q 1990 Elsevier Scientific Publishers Ireland, Ltd.

1A

9/13

9/13

40/350

8

Tumour

25%

43%

30%

50%

17/20

2/20

10 b 12

tissue data

2/20 4/20

6/20 9/20 4/20 2/20

S/20

18/20

17

4/50 3/50

4,‘50 6/50

9/50 10/50 9/50 13/50 6/50 3/50

31/50

18

4/50 3/50

6,‘70 lo/70

17/50 10/50 17/90 22/70 lo/70 5/70

49/70

Total

9/54

22/54

1

26/54

15,‘20

2

Serum/plasma

26/58

3

data

20,‘66

4

16,‘41

48,‘75

22/75

5

36/49

26/68

7

76/135

9

18/61 29/61

11

13/24

13

15/63

15

18/61 74/222

208/367

117/308

Total

a In patient serum. b No. of patients not stated. POMC: pro-opiomelanocortin; GRF: gastrin releasing factor; CGRP: calcitonin gene-related peptide; VIP: vaso-intestinal peptide; ADH: antidiuretic hormone; LH: luteinizing hormone; ME-AGL: methionine-enkephahn arginine-glycine-leucine; NPY: neuropeptide tyrosine. References: 1. Greco et al., 1981; 2. Gropp et al., 1980; 3. Gropp et al., 1981; 4. Gropp et al., 1982; 5. Hansen et al., 1980; 6. Havemann et al., 1985; 7. Krauss et al., 1981; 8. List et al., 1986; 9. Luster et al., 1982; 10. Luster et al., 1985; 11. North et al., 1980; 12. Noseda et al., 1987; 13. Ode11 et al., 1979; 14. Pettengill et al., 1985; 15. Ratcliffe et al., 1982; 16. Sorenson et al., 1981; 17. Yamaguchi et al., 1985; 18. Yamaguchi et al., 1986.

3/18

2/18 9/18

l/13 5/13

l/5 4/5

2/13

14/18

lo/13

4/5

l/5

4/5

Total

BY SCUCL

4/5

16

Cell line data

PRODUCED

Ref. 14

Bombesin POMC derived GRF Somatostatin Calcitonin CGRP VIP Oxytocin ADH Neurotensin LH Motilin a ME-AGL NPY

Peptide

PEPTIDES

TABLE

TABLE 1B HORMONES

PRODUCED

BY NON-PULMONARY

Tissue of origin

Bombesin

Bladder Colon Endometrium Gall bladder

l/l l/l

Larynx Minor salivary gland Oesophagus Pancreas Prostate

l/l

Paranasal sinuses Stomach Thymus

SCUCs

POMC derived

l/l l/19

ss

Other

b VIP 2/21

l/22 l/l

CT

a

l/J l/l a 9/17 b 3/3 b l/4 l/2 5/22

l/l

2/4 l/2

Ref. 9 9 7 1 9 3 9 2 4 5 10 6 9 8

’ Cell line data. b Patient serum/plasma. SS: somatostatm; CT; calcitonin; VIP: vase-intestinal peptide. References: 1. Albores-Saavedra et al., 1982; 2. Corrin et al., 1973; 3. Hayashi et al., 1987; 4. Jelbart et al., 1988a; 5. Jelbart et al., 1988b; 6. Kameya et al., 1980; 7. Olson et al., 1982; 8. Sayler et al., 1976; 9. Springah et al., 1986; 10. Wenk et al., 1977.

detailed discussion pertaining to each of these hormones would be outside the scope of this review. For this reason, we will limit further discussion to BN/GRP, given its reported role in the pathophysiology of SCUCL, and to calcitonin and POMC, the precursor of ACTH, since their production has been widely reported in both pulmonary and non-pulmonary SCUC. Possible autocrine regulation of SCUC growth by ectopic hormone production Studies of SCUCL have revealed that some tumours and cell lines which contain high concentrations of BN/GRP also express specific, saturable high-affinity receptors for these peptides (Moody et al., 1983, 1985). Similarly, two SCUCL cell lines, NCI-H146 and NCI-H187, shown to contain 0.2 and 1.2 pmol/mg protein of low molecular weight opioid immunoreactivity, expressed 50-100 fmol/mg protein of specific, high affinity (Kd 0.9 and 1.2 nM, respectively), membrane receptors for opioids (Roth and Barchas, 1986). The expression of both hormone and receptor by the same SCUC cells may be coincidental,

but may reflect an autocrine role for the hormone in the regulation of growth of the tumours. Such an autocrine mechanism has been substantiated for BN/GRP in SCUCL (Carney et al., 1983, 1987; Gaudino et al., 1988) as discussed later in this review. One study has shown that ACTH produced by a SCUCL induces an increased growth rate of the tumour in vitro (Luster et al., 1985) suggesting a potential autocrine role for ACTH in this case. The regulation of cell growth of SCUCL GRP

by BN/

Several laboratories have demonstrated that the majority of human SCUCL produce BN, and BN-like peptides, such as GRP (Moody et al., 1981; Wood et al., 1981; Erisman et al., 1982; Yamaguchi et al., 1983, 1986; Sorenson et al., 1985), and these have been proposed as possible autocrine growth factors for SCUCL (Camey et al., 1983, 1987). However, only 5% of patients with SCUCL have elevated plasma levels of BN, and this has limited its value as a biological marker for these tumours (Pert and Schumacher, 1982). A

4

xenograft line developed from a human small cell carcinoma of the prostate, UCRU-PR-2, was also found to contain BN in early passage (Jelbart et al., 1988a), but these findings could not be substantiated in later passage xenografts (Jelbart et al., 1989) suggesting that BN production by the tumour was not required for its growth as a xenografted line. Bombesin is a 14 amino acid peptide first isolated from the skin of a European ampbibian, Bombina bombina (Anastasi, 1971). Its mammalian equivalent, GRP, a 27 amino acid peptide with a carboxy-terminal heptapeptide sequence identical to that of BN, is found predominantly in the brain (Moody et al., 1978; Walsh et al., 1978), lung (Wharton et al., 1978; Yamaguchi et al., 1983) and gastrointestinal tract (McDonald et al., 1978; Walsh et al., 1978). Given their similar pharmacological effects in laboratory animals, and since they both bind to a single class of high affinity receptors (found in brain, pancreas and pituitary cells), BN and GRP are referred to as BN/GRP peptides (Carney et al., 1987). The binding of BN/GRP to appropriate cells stimulates hormone production by these cells (reviewed in Erspamer, 1980; Jensen et al., 1984). The role of these peptides as possible autocrine stimulators of SCUCL is not unexpected, since BN/GRP has been mooted as a putative growth stimulatory agent for normal fetal lung and gut. High levels are found in fetal and newborn, but not in adult, lung (Wharton et al., 1978; Yamaguchi et al., 1983); in vivo administration of BN induces gastric hyperplasia in rats (Lehy et al., 1983) and pancreatic hyperplasia in mice (Alexander et al., 1988). BN and GRP stimulate clonal growth and DNA synthesis of lung small cell carcinoma cell lines in vitro (Cuttitta et al., 1985; Weber et al., 1985; Carney et al., 1987) with an optimum concentration of 50 nM (BN). When high doses are used, suppression of clonal growth occurs, suggesting down-regulation of receptor expression by high levels of the ligand. In contrast, exogenous bombesin did not stimulate growth of all SCUCL cell Iines in liquid culture assay (Layton et al., 1988). Mitogenic effects of BN/GRP have been observed in Swiss 3T3 cells (Zachary and Rozengurt, 1985). Specific BN/GRP receptor-binding activity has been reported in tumour-derived cell

lines of SCUCL (Moody et al., 1983,1985) and in Swiss 3T3 cells (Zachary and Rozengurt, 1985; Cirillo et al., 1986). A monoclonal antibody raised against the binding moiety of BN/GRP prevents growth of tumour cells in vitro (Cuttitta et al., 1985) and of transplanted human tumour xenografts in nude mice (Cuttitta et al., 1985; Alexander et al., 1988). Similarly, a synthetic peptide, [D-&g’, D-Proz, DP (spantide), which acts as -T 7*93 Leu’rjsubstance a BN receptor antagonist (Jensen et al., 1984), inhibits the mitogenic effects of BN/GRP on Swiss 3T3 cells in vitro (Corps et al., 1985; Zachary and Rozengurt, 1985) raising the possibility that antagonists for peptides of the bombesin family could be of importance for future approaches to the clinical treatment of SCUCL. However, the effects of spantide could be overcome by sufficiently high concentrations of bombesin in vitro (Jensen et al., 1984), and the peptide failed to inhibit gastrin secretion in dogs (Pappas et al., 1985) suggesting that it was not sufficiently potent for in vivo use. More recently, a new generation of bombesin/BRP antagonists, based on reduction of different peptide bonds (for example, 9-10, 13-14) in Leu-bombesin, rather than on the classical side chain modification strategies, has been developed (Coy et al., 1988). These novel compounds exhibit physical properties almost identical to bombesin and appear to be receptor specific showing competitive i~bition of both bombesin-stimulated amylase release from guinea pig pancreatic acini, and bombesin-stimulated growth of murine Swiss 3T3 cells (Coy et al., 1988; Woll et al., 1988), in the latter case, with an IC,, of 18 nM. The authors propose that conformational changes introduced into bombesin by 9-10 or 13-14 peptide bond replacements lead to alterations in hydrogen bonding and loss of biological activity. The development of a bombesin receptor antagonist with useful therapeutic properties may require additional synthetic work aimed at improving receptor affinity even further, and particularly at improving in vivo pharmacokinetic properties. Similar studies using analogues of somatostatin-14 have shown marked inhibition of human small cell lung carcinoma growth both in vitro and in vivo (Taylor et al., 1988a, b).

5

The gene for prepro-gas&h-releasing

peptide

In mammalian cells, BN/GRP is produced from a precursor molecule, prepro-gastrin-releasing peptide; cDNA clones for this peptide have been obtained from a pulmonary carcinoid tumour (Spindel et al., 1984). This gene encodes a putative pro-GRP molecule consisting of a signal sequence (amino acids -23 to -l), GRP itself (amino acids l-27) and a GRP-associated peptide (Cterminal peptide, amino acids 31-125) the function of which is not yet known. Different forms of the prepro-GRP gene have been found. Two types were detected in a cDNA library from a lung carcinoid tumour (Spindel et al., 1986), which differ in the presence of a 19 base insertion in the sequence coding for the 3’-GRPassociated peptide, occurring after amino acid 98 in pro-GRP. Similarly, two peaks of immunoreactive GRP have been revealed in both fetal lung and lung tumour tissue (Yamaguchi et al., 1983). The ratio of these two peaks differs from tumour to tumour. Recently, both of these mRNA species plus a third type of prepro-GRP messenger RNA have been found in five of nine cell lines or metastatic deposits of SCUCL (Sausville et al., 1986). These three types of mRNA are all derived from a single transcript by alternative splicing (Sausville et al., 1986). The production of pro-GRP appears to have major significance in SCUCL. Antibodies prepared against the putative C-terminal peptide of pro-GRP gave strong, diffuse, positive cytoplasmic staining in 70% of small cell carcinomas (175/250), 63% of atypical carcinoid of the lung (31/49) and 16% of carcinoid of the lung, in contrast to BN immunostaining which was focal and present in 35% of carcinoids and only 25% SCUCL and 22% of atypical carcinoids (Hamid et al., 1987). The expression of the C-terminal peptide, but not BN, appeared to correlate with increased malignancy and shortened life expectancy. The patterns of staining suggested that in tumour tissue pro-GRP can be synthesized and released without packaging, whereas in normal tissues BN has first to be packaged into neurosecretory granules for storage (Hamid et al., 1987). In a similar study, Yamaguchi and coworkers (1983) used a radioimmunoassay (RIA) specific

for the carboxy-terminal portion of immunoreactive GRP and demonstrated that fetal lung and Ir-GRP-producing tumours contained no measurable Ir-BN. These authors proposed that the lack of immunoreactivity of SCUCL with BN could be due to scarcity of secretory granules in this type of tumour or to non-recognition of the molecular forms of the active hormone produced by the tumour cells. The 21 amino acid fragment of the C-terminal peptide used to raise antiserum is in the conserved part of the molecule which should be common to most SCUC. Further analysis revealed that the RIA used to measure BN recognized the glutamine residue at position 8 of BN and did not cross-react with pro-GRP which contains a histidine in this position. This indicates that the active Ir-GRP found in fetal lung and in primary lung tumours has an amino acid sequence similar to GRP, rather than to BN (Yamaguchi et al., 1983). Mode of action of BN/ GRP Binding of BN or related substances to cell lines derived from SCUCL results in a rapid and transient increase in intracellular calcium concentration (Heikkila et al., 1987). This ‘is inhibited by modification of the carboxyl-terminal octapeptide of BN, or by a monoclonal antibody which binds to BN. In Swiss 3T3 mouse fibroblasts, high affinity receptor binding of BN is associated with a tyrosine-kinase activity (Cirillo et al., 1986; Zachary and Rozengurt, 1986) which phosphorylates a 115 kDa protein (~115). The human counterpart of ~115 has been demonstrated in SCUCL (Gaudino et al., 1988) and is phosphorylated in the absence of exogenous BN/GRP, suggesting an autocrine role for BN/GRP in these cells. Regulation of bombesin / GRP and secretion in cell cultures of SCUCL

calcitonin

Only one group appears to have addressed exogenous regulation of the secretion of BN/GRP by SCUCL (Sorenson et al., 1985). These findings are summarized in Table 2. Cholinergic and adrenergic agonists and dopamine stimulated BN/GRP release; histamine and acetylcholine appeared to act on cell surface receptors of the

6 TABLE

2

MODULATION

OF SECRETION

OF BN/GRP

Substance

AND CALCITONIN ~n~ntration

Dibutywl-CAMP Choline&c agonists

NR IO-‘M

b, e.g. bethanechol

50400

Adrenergic agonists Dopamine Histamine Acetylcholine Carbachol Cholecystokii Gastrin

NR NR NR NR NR 1O-3 M 5~10~~ M 5x10-‘M 1O-6 M lo-‘M lo-20 units/litre 1O-6 M lo-‘M 1.8-3.6 x 10K5 M

Secretin Vasoactive Arginine

intestinal

peptide

vasopressin

NR: not reported. a After Sorenson et al., 1985. b Choline& receptors on SCUCL type (Sorenson et al., 1985).

are of muscarinic

PM

type (Sorenson

H,-~sta~~~r~c, and muscarinic type respectively, to increase hormone production. Secretion was also elevated by increasing CAMP levels, indicating that this is part of the sequence of metabolic mechanisms involved in the secretory process in neoplastic cells, as has been observed in many normal endocrine cells. A series of hormones including cholecystokinin, gastrin, glucagon, secretin, somatostatin, thyroid-releasing hormone, vasoactive intestinal peptide and a&nine vasopressin failed to affect DMS53 cell secretion of BN,‘GRP. Production of pro-opiomelanoeortin rived peptides by SCUC

SCUCLa

Levels of secretion CT

BN,‘GRP

r

f f

T (small MW)

Chloroquine

Glucagon

BY CULTURED

(POMC)-de-

POMC is one of three opioid neuropeptide precursors sharing similar genomic and mRNA organisation (Imura et al., 1985). It is normally synthesized predominantly in the anterior (AP) and neuro-intermediate lobes of the pituitary (NIL), but is also found in the h~oth~amus and

1 (large MW)

NR

f t r f t NR NR NR NR NR NR NR NR NR

T t t T

et al., 1983) whilst histamine

T _ _ _ _ _ _ _ _

receptors

are of H,-histaminergic

in peripheral tissues, including the thyroid, pancreas, male and female reproductive organs, gastrointestinal tract, immune system, adrenal gland and lung. In each tissue, variable precursor cleavage and processing events result in a pattern of end product combinations, and thus with the possibility of unique biochemical and physiological consequences (Dennis et al., 1983; de Wied et al., 1987). To date, ACTH appears to be the most common ectopic hormone in SCUC, sometimes in association with clinically evident Cushing’s syndrome (Strott et al., 1968; Jelbart et al., 1988b). However, unlike studies of peptide production by normal tissues, analysis of the molecular forms of ectopic ACTH and P-lipotrophic hormone production (/3-LPH) by SCUC has received scant attention. Studies of the production of POMC-derived peptides by SCUC are summarized in Table 3. In both SCUC of the lung and prostate, POMC undergoes proteolytic cleavage to P-LPH and ACTH and subsequently to the major end prod-

TABLE

3

POMC-DERIVED

PEPTIDES

PRODUCED

BY SMALL CELL CARCINOMAS

SCLC (patient serum + cell culture supematants)

SCUCP (xenograft homogenates, cell culture supematants)

Other tumours

ACTH which is also processed to a-MSH and @-MSH

ACTH(l-39) processed to a-MSH and CLIP Diacetyl a-MSH predominates, but only in low concentration (5)

Stomach: ACTH detected (1,ll)

(2,3,4,6,7,8,9,10,12) /3-LPH p-EP

present which is processed

to

Pancreas: ACTH detected (1,ll)

fi-EP(l-27) predominates with very little N-acetylation (5)

(2,3,6,7,8,9) References: 1. Corrin et al., 1973; 2. Gropp et al., 1981; 3. Hansen et al., 1980; 4. Hirata et al., 1975; 5. Jelbart et al., 1988a; 6. Krauss et al., 1981; 7. Mackay et al., 1977; 8. Ode11 et al., 1979; 9. Ratchffe et al., 1982; 10. Sorenson et al, 1981; 11. Springall et al., 1986; 12. Yamagucbi et al., -1985.

ucts, @-endorphin (@-EP), cw-melanocyte-stimulating hormone (ar-MSH) and corticotrophin-like immunor~ctive peptide (CLIP). High performance liquid chromatography (HPLC) analysis has indicated that in one SCUCP, the predominant p-endorphin is the P-EP(l-27) species (O’Mara et al., unpublished); this species is only one of several forms found at significant levels in the NIL. Nacetylation of the endorphin, which has not been reported in SCUCL, is detectable in SCUCP, although to a much lesser extent than occurs in the NIL or in peripheral tissues, such as pancreas and thyroid (see Smith and Funder, 1988). Acetylation of a-MSH is also evident in SCUCP, as in the NIL (rat), pancreas (rat), male reproductive tract (rat) and adrenal medulla (human) (Smith and Funder, 1988), but has not been reported in SCUCL. The pattern of fi-LPH and ACTH processing in SCUCL and SCUCP appears to reflect that reported in rat hypothalamus (Emson et al., 1984) except for further cleavage of B-EP. In contrast, POMC processing in SCUC differs from that seen in the pituitary, and in the peripheral tissues of various species. The reasons for different processing of the POMC gene in tumour cells compared with normal cells are not known. One possibility is that gene regulation may be altered following chromosomal or gross DNA rearrangements within neoplastic tissues or mutations within the gene promoter. The POMC gene is located on the short arm of chromosome 2 (Shows et al.,

1982); the SCUCP line, UCRU-PR-2, has a marker on chromosome 2 (Pittman et al., 1987). However, in situ hyb~dization studies to determine whether this marker is associated with the POMC gene are yet to be carried out. Karyotypic analyses have shown that many SCUCL carry a deletion within the short arm of chromosome (3p14-~23) (Naylor et al., 1987), the site of a putative anti-oncogene or suppressor gene. This deletion also overlaps the region 3~21-3~25 which is the location of the erbA /i’ (thyroid hormone receptor) sequence, one of a superfamily of regulatory genes coding for hormone receptors. At least one copy of this gene was also deleted in 6/6 SCUCL cell lines or tumours studied (Dobrovic et al., 1988). The relevance of the 3p deletion to ectopic hormone production by SCUCL has not been defined, and this deletion was not seen in a line of SCUCP (Pittman et al., 1987).

Aitemative

splicing of mRNA for POMC

The size of the messenger RNA for POMC is also relevant to the biological activity and pattern of the resulting peptides. Three different transcripts, 800 bp, 1200 bp and 1400 bp, have been detected in different tissues. The 1200 bp mRNA transcript found in both lobes of the pituitary is actively transcribed to yield biologically active peptides (Jeanotte et al., 1987b); the 1400 bp transcript has been described in the hypothalamus

8

and appears to result from increased polyadenylation (Jeanotte et al., 1987a); the 800 bp transcript results from initiation of transcription at a site under the \;ontrol of the GC box promoter sequence at the 3’ end of exon 2. The 5’ truncated message is thought not to yield active peptides, since it lacks exon 1 and exon 2, which code for the signal peptide for membrane translocation to the endoplasmic reticulum and for y-MSH (Lacaze-Masmonteil et al., 1987). The 800 bp transcript is the predominant form of POMC mRNA found in ovary, testis and placenta (Pintar et al., 1984; Chen et al., 19861987; de Bold et al., 1988; Ivell et al., 1988). By the use of sensitive Slnuclease mapping, however, small amounts of the 1200 bp mRNA have been located in the testis, where it is thought to be responsible for the production of all of the POMC peptides (LacazeMasmonteil et al., 1987). Both normal length (1100 base) and large (1300 or 1450 base) POMC sequences have been described in thymic carcinoids and their metastases (Tsukada et al., 1981; de Bold et al., 1983; de Keyzer et al., 1985). In vitro translation of the mRNAs from a thymic carcinoma indicated that the longer mRNA did not contain additional amino acid coding sequence (Tsukada et al., 1981). DNA sequencing of a POMC cDNA derived from both a thymic carcinoid (de Bold et al., 1983) and from a metastasis of thyroid carcinoma (Steenbergh et al., 1984) indicated that the 3’ end of the mRNA corresponded to the normal human gene sequence. More recently, a study of POMC mRNA in nine human ACTH-secreting tumours including five pulmonary carcinoids (Clark et al., 1989), showed that in most cases the POMC mRNA was similar to or slightly larger than that in the normal pituitary. However, two pancreatic tumours expressed 800, or 700 and 900 base sequences, and a 1500 base mRNA variant was found in low levels in two cases, and as the principal mRNA in one. In one case, this was because of a longer poly A tail. Sl nuclease and primer extension studies were undertaken to investigate whether an alternative transcriptional start site was functional. In most cases, analysis indicated that the tumour transcripts originated from the conventional promoter. A minor start site at -734 was unique to the ectopic ACTH-tumour RNAs, whereas other

minor start sites (- 83, - 88 and - 94) were found to a degree in ‘normal’ pituitary RNA.

Regulation carcinomas

of POMC

production

in small

cell

In contrast to numerous studies showing differential regulation of POMC production in the pituitary and in peripheral tissues (for reviews, see Herz and Millan, 1985; Smith and Funder, 1988), the physiolo~~l or molecular regulation of POMC production by SCUC has been little studied. Treatment of malignant cells from SCUCL (Hirata et al., 1975) or from carcinoid of the stomach (Suda et al., 1986) with corticotrophin-releasing hormone/corti~otrophin-releasing factor (CRH/ CRF), or with CAMP, increased secretion of POMC-derived peptides. In this respect these tumours behaved like the pituitary, where CRF and CAMP have been shown to regulate POMCderived peptide secretion (Labrie et al., 1971; Abou-Samra et al., 1986).

Calcitonin CT and calcitonin gene-related peptide (CGRP) are two peptides commonly produced by SCUC (see Table l), especially of the lung. CT expression has also been identified in other neoplasms including medullary thyroid carcinoma (Steenbergh et al., 1984; Nakagawa et al., 1987), acute myeloid and lymphoid malignancies (Baylin et al., 1987; Pfluger et al., 1988) and Ewing’s sarcoma (Hoppener et al., 1987, 1988). In normal tissue, CT, CGRP, and katacalcin (KT), a family of closely related peptides, are found in the thyroid, and in the case of CGRP, in the central nervous system (CNS). Calcitonin and KT are secreted by thyroid cells in equimolar amounts (Hillyard et al., 1983) due to cleavage of a common precursor. CT (32 amino acids) appears to inhibit osteoclast activity (Chambers et al., 1983, 1985a, b; Chambers and Moore, 1983) and chronic CT treatment of Paget’s disease reduces osteoclast numbers (Macfntyre et al., 1987). In contrast, the physiological function of the C-terminal peptide, KT (21 amino acids), is as yet unknown (Maclntyre et al., 1987).

9

CGRP (37 amino acids) has been shown to be involved in calcium metabolism and is a potent vasodilator (MacIntyre et al., 1987). CGRP can also act as a neurotransmitter or neuromodulator centrally, but when released from perivascular nerve terminals or from motor neurons, it modulates anterior tone or is thought to regulate the muscle acetylcholine receptor state, respectively (Zaidi et al., 1987). Differential

splicing of calcitonin genes

Two identified, functional calcitonin genes, CALC-I and CALC-II, and a postulated pseudogene (CALC-III), are located on the short arm of chromosome 11 (MacIntyre et al., 1987; Hoppener et al., 1988). CALC-I and CALC-II each contain 6 exons. The CALC-I gene encodes CT, KT and CGRP-I, whilst CALC-II encodes only CGRP-II. There is 92% homology between the CGRP-I-encoding exon 5 of the CALC-I gene and the CGRP-II-encoding exon 5 of the CALC-II gene, resulting in 3 amino acid differences between the two peptides. Tissue-specific, differential splicing of the CALC-I gene product, containing two polyadenylation sites (after exons IV and VI, respectively (Amara et al., 1984)), is responsible for the expression of the two related peptides, CT and CGRP, from the same gene (Jonas et al., 1985; MacIntyre et al., 1987). Exon I, common to both mRNA species, is non-coding; exons II and III encode the common 75 amino acid N-terminal peptide; exon IV encodes CT; exons V and VI are CGRP coding and CGRP non-coding, respectively (Hoppener et al., 1987; MacIntyre et al., 1987). The sequence of events in the differential splicing of the CT/CGRP gene transcript has been proposed by Bovenberg and coworkers (1986). Following splicing and translation of the mRNA, the resulting prepeptides are prot~lytically cleaved to release the functional peptides, CT and CGRP (Hoppener et al., 1987; MacIntyre et al., 1987). In transgenic mice, most cells not expressing endogenous CT/CGRP from a metallothioneinCT fusion gene have a clear splicing choice for the CT or CGRP mRNA, resulting in 90-97% CT mRNA in most tissues, and CGRP transcripts in neuronal tissue (Crenshaw et al., 1987).

Hy~~e~yiation

of CT genes

A ‘hot spot’ for DNA hypermethylation near the CT genes on the short arm of chromosome 11 has been identified in human malignancies (de Bustros et al., 1988). Recently, hypermethylation of CCGG sites of the 5’ region of the calcitonin gene has been demonstrated in SCUCL, lymphomas and leukaemias (Baylin et al., 1986, 1987) and in colonic adenomas (13/19), carcinomas (4/l 3) and established colon carcinoma cell lines (18/19) (Silverman et al., 1989), despite the presence of overall genomic DNA h~omethylation in these neoplasms. The significance of this finding in terms of ectopic hormone production by these tumours has not been addressed. Relation

of CT production by ~0~

cells

Sorenson and coworkers (1985) have reported studies of the effects of various exogenous hormones or ligands on CT secretion by SCUCL cells (summarized in Table 2). Three different molecular forms of CT were produced by cultured cells, and these were modulated both quantitatively and qualitatively by cholinergic agents. Bethanechol caused an increase in overall CT secretion, predominantly in the 8000 molecular weight (MW) form. In contrast, chloroquine down-modulated secretion, mostly of the 18,000 MW form. Increased doses of chloroquine resulted in an increased relative amount of the smaller MW form of CT, but not above baseline levels. Like BN/GRP, CT secretion was enhanced by choline@, adrenergic, dopaminergic and histaminergic ligands and by CAMP. CT,/CGRP expression and release could be differentially regulated in the rat 44-2C cell line (Zeytin et al., 1987); both calcium and dexamethasone treatments were found to decrease CT content and release, and to diminish the levels of CT- and CGRP-specific mRNA levels. Despite these studies, the importance of the production by CT/CGRP by tumours to the biology of the tumours has not been studied. Conclusions Ectopic hormone production by small cell undifferentiated carcinomas is of considerable significance, since it can be associated not only with

10

clinical features, such as Cushing’s syndrome, but also with autocrine control of tumour cell growth (as in the case of BNfGRP, and possibly ACTH). The production of appropriate inhibitors or monoclonal antibodies could therefore in theory be used to specifically modify tumour growth. This would be very advantageous, given the poor prognosis of patients with SCUC. The design of improved, conformationally restricted analogues for bombesin has recently been initiated (Coy et al., 1988). There is a paucity of studies on the regulation of the production of ectopic hormones by SCUC; such studies have suggested that the regulation differs from that which OCGU~S in normal tissue, especially in the case of PUMC production. It is not clear whether the mechanisms involved in these differences in regulation are the same for SCUC of different organs, or whether they differ from one tissue to another. The differences between regulation of ectopic hormones by neoplastic as compared with normal tissue may relate to the deletion of crucial regulatory elements, or suppressor genes, or to unusual methylation patterns of the genes of interest, as has been described for CT in SCUCL. It is of interest that alternative splicing of mRNA occurs for several of the precursor proteins involved in the production of ectopic peptides by SCUC, including prepro-GRP (Sausville et al., 1986; Spindel et al., 1986), calcitonin (Amara et al., 1982), and POMC (Oates and Herbert, 1984). The heterogeneity in POMC mRNAs does not appear to result in different POMC proteins, and the biological significance of the alternative splicing in this case remains uncertain. More detailed studies are required to explain the mechanisms by which ectopic hormone production is regulated in SCUC. Acknawkdgement P.J. Russell was supported by a grant from the National Health and Medical Research Council, Australia. References Abou-Samra, crinoiogy

A.B., C&t, K.J. and Aguilera, 118. 212-217.

G. (1986) Endo-

Albores-Saavedra, J., Soriano, J., Larraza-Hernandez, 0.. Aguirre, J. and Henson, D.E. (1984) Human Path&. 15, 639-646. Alexander, R.W., Upp, Jr., J.R., Poston. G.J., Gupta, V., Townsend, Jr., CM. and Thompson, XC. (1988) Cancer Res. 48,439~441. Almagro, U.A. (1985) Cancer 55, 608-614. Amara, S.C., jonas, V., Rosenfield, M.G., Ong. ES. and Evans, R.M. (1982) Nature 298, 240-244. Amara, S.G., Evans, R.N. and Rosenfeld, M.G. (1984) Mol. Cell. Biol. 4, 2151-2160. Anastasi, A., Erspamer. V. and Bucci, M. (1971) Experienta 27, 166-167. Baylin, S.B., Hoppenet, J.W.M., de Bustros, A., Steenbergh, P.H.D., Lips, C.J.M. and Nelkin, B.D. (1986) Cancer Res. 46, 2917-2922. Bay& S.B., Fearon, E.R., Vogelstein. B., de Bustros, A., Sharkis. S.J., Burke, P.J., Staaf, S.P. and Nelkin, B.D. (1987) Blood 70, 412-417. Bovenberg, R.A., Adena, G.J.. Jansz, H.S. and Baas, P.O. (1988) Nucleic Acid Res. 16, 7867-7883. Camey, D.N., Cuttitta, F.C., Gazdar, A.F. and Minna, J.D. (1983) Proc. Am. Sot. Chn. Oncol. 2, 14 (abstract). Carney, D.N., Cuttitta, F., Moody, T.W. and Minna, J.D. (1987) Cancer Res. 47, 821-825. Chambers, T.J. and Moore, A. (1983) J. Clin. Endocrinol. Metab. $7, 819824. Chambers, T.J., Athanasou, N.A. and FuBer, K. (1984) J. Endocrinol. 102, 281-286. Chambers, T.J., Fuller, K., McSheehy, P.M.J. and Pringfe. J.A.S. (198Sa) J. Pathol. 145, 297-305. Chambers. T.J., McSheehy, f,M.J., Thomson, B.M. and Fuffer, K. (198Sb) Endocrinology 116234-239. Chen, C.-L. and Madigan, M.B. (1987) Endocrinology 121, 590-596. Chen, C.-L., Chang, C.-C., Krieger, D. and Bardin, C.W. (1986) Endocrinology 118,2382-2389. Cirillo, D., Gaudino, G., Naldini, L. and Comoglio, P.M. (1986) Mol. Cell. Biol. 6, 4641-4649. Clark, A.J.L., Lavender, P.M., Besser, G.M. and Rees, L.H. (1989) J. Mol. Endocrinol. 2, 3-9. Corps, A.N., Rees, L.H. and Brown, K.D. (1985) Biochem. J. 231, 781-784. Corrin, B., Giiby, ED., Jones, N.F. and Patrick, J. (1973) Cancer 31, 1523-1527. Coy, D.H., Heinz-E&m, P., Jiang, N.-Y., Sasaki, Y., TayIor, J., Moreau, J.-P., Wolfrey, W.T.. Gardner, J.D. and Jensen, R.T. (1988) J. Biol. Chem. 263, 5056-5060. Crenshaw, E.B., Russo, A.F., Swanson, L.H. and Rosenfeeld, M.G. (1987) Cell 49, 389-398. Cuttitta, F., Camey, D.N., Mulshine, J., Moody, T.W., Fedorko, J., Fischler. A. and Minna, J.D. (1985) Nature 316, 823-826. de Bold, CR., Schworer, ME., Connor, T.B., Bird, R.E. and Orth, D.N. (1983) Science 220, 721-723. de Bold, CR., Nicholson, W.E. and Orth, D.N. (1988) Endocrinology 122, 2648-2657. de Bustros, A., Nelkin, B.D., Silverman, A., Ehrlicb G., Poiesz, B. and Baybn, S.B. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 569335697.

11 deKeyzer, Y., Bertagna, X., Lerme, F., Girard, F., Luton, J.-P. and Kahn, A. (1985) J. Clin. Invest. 76, 1892-1898. Dennis, L., Lazure, C., Seidah, N.G. and Chretien, M. (1983) J. Cbromatogr. 266, 163. de Wied, D. and de Kloet, E.R. (1987) Ann. N.Y. Acad. Sci. 512, 328-337. Dobrovic, A., Houle, B., Belouchi, A. and Bradley, W.E.C. (1988) Cancer Res. 48, 682-685. Emson, PC, Corder, R., Ratter, S.J., Tomlin, S., Lowry, P.J., Rees, L.H., Arregui, A. and Rosser, M.N. (1984) Neuroendocrinology 38,45-50. E&man, M.D., Linnoila, RI., Hemandez. O., di Augustine, R.P. and Lazarus, L.H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2379-2383. Erspamer, V. (1980) in Gastrointestinal Hormones (Glass, G.B.J., ed.), Raven Press, New York. Gaudino, G., Cirillo, D., Naldini, L., Ross%, P. and Comoglio, P.M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 21662170. Giraud, A.S., Smith, LA. and Rundle, SE. (1987) Mol. Cell. Endocrinol. 53, 245-250. Greco, A.F., Hainsworth, J. and Sismann, A. (1981) in Small Cell Lung Cancer (Greco, A.F., Oldham, R.K. and Bunn, P.A., eds.), pp. 177-224, Grune and Stratton, New York. Gropp, C., Havemann, K. and Scheuer, A. (1980) Cancer 46, 347-354. Gropp, C., Luster, W., Havemann, K. and Lehman, F.G. (1981) in CEA und andere Tumor Marker (Uhlenbmck, G., and Winzter, G., eds.), pp. 358-363, Tumor Diagnostik, Leonberg. Gropp, C., Havemann, K., Kalbfleixh, H. and Luster, W. (1982) Dtsch. Med. Wochenschr. 107, 977-980. Hamid, Q.A., Addis, B.J., Springall, D.R., Ibrahim, N.B.N., Ghatei, M.A., Bloom, S.R. and Polak, J.M. (1987) Virchows Arch. Abt. A Pathol. Anat. Histopathol. 411, 185-192. Hansen, M., Hammer, M. and Hummer, L. (1980) Cancer 46, 2062-2067. Havemann, K., Luster, W., Gropp, C. and Holle, R. (1985) Recent Results Cancer Res. 97, 65-76. Hayasbi, Y., Nagamine, S., Yanagawa, T., Yoshida, H., Yura, Y., Azuma, M. and Sata, M. (1987) Cancer 60, 1583-1588. Heikkila, R., Trepel, J.B., Cuttitta, F., Necker, L.M. and Sausville, E.A. (1987) J. Bid. Chem. 262, 16456-16460. Hen, A. and Millan, M.J. (1985) in Opioids Past, Present and Future: Symposium to Celebrate Eightieth Birthday of H.W. Kosterlitz (Hughes, J. et al., eds.), Chapt. 7, Taylor and Francis, London, Philadelphia, PA. Hillyard, J.C., Myers, C., Abeyasekera, G., Stevenson, J.C., Craig, R.K. and MacIntyre, I. (1983) Lancet i, 846. Hirata, Y., Yamamoto, H., Matsukura, S. and Imura, H. (1975) J. Clin. Endocrinol. Metab. 41, 106-114. Hirsch, M.D. and O’Donohue, T.L. (1985) Peptides 6,293-296. Hoppener, J.W.M., Steenbergh, P.H., Siebos, R.J.C., Visser, A., Lips, C.J.M., Jansz, H.S., Bechet, J.M., Lenoir, G.M., Born, W., Haller-Brem. S., Petermann, J.B. and Fischer, J.A. (1987) J. Clin. Endocrinol. Metab. 64, 809-817. Hoppener, J.W.M., Steenbergh, P.H., Zandberg, J., Adema, G.J., Geurts van Kessel, A.H., Lips. C.J.M. and Jansz, H.S. (1988) FEBS Lett. 233, 57-63.

Imura, H., Kato, Y., Nakai, Y.. Nakao, K., Tanaka, I., Jingami, H., Koh, T., Yoshimasa, Y., Tsukada, T., Suda, M., Sakamoto, M., Morii, N., Takahashi, H., Tojo, K. and Sugawara, A. (1985) J. Endocrinol. 107, 147-157. Ivell, R., Walther, N. and Morley, S. (1988) Nucleic Acids Res. 16, 7747. Jeanotte, L.. Burbach, J.P.H. and Drouin, J. (1987a) Mol. Endocrinol. 1, 749-757. Jeanotte, L., Trifiro, M.A.S., Plante, R.K., Chamberland, M. and Drouin, J. (1987b) Mol. Cell. Biol. 7, 4058-4064. Jelbart, M.E., Russell, P.J., Fullerton, M., Russell, P., Funder, J.W. and Raghavan, D. (1988a) Mol. Cell. Endrocrinol. 55, 167-172. Jelbart, M.E., Russell, P.J., Russell, P. and Raghaven, D. (1988b) in Textbook of Uncommon Cancer (Williams, C-J., Krikorian, J.G., Green, M.R. and Raghavan, D., eds.), pp. 49-262, John Wiley and Sons, London. Jelbart, M.E., Russell, P.J., Russell, P., Wass, J.. Fullerton, M., Wills, E.J. and Raghavan, D. (1989) Prostate 14, 163-175. Jensen, R.T., Jones, S.W., Folkers, K. and Gardner. J.D. (1984) Nature 309, 61-63. Jonas, V., Lin, C.R., Kawashima, E., Semon, D., Swanson, L.W., Mermod, J.-J., Evans, R.M. and Rosenfeld, M.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1994-1998. Kameya, T., Shimosato, Y., Ada&i, I., Abe, K., Ebihara, S. and Ono, I. (1980) Cancer 45, 330-339. Kendall, J.W., 01~011, E. and Gaudette, D. (1980) in Proceedings of the VI Intemational Congress of Endocrinology, Melbourne, p. 528 (abstract 637). Krauss, S., Macy, S. and Ichiki, A.T. (1981) Cancer 47, 24852492. Labrie, F., Giguere, V., Meunier, H., Simard, J., Gossard, F. and Raymond, V. (1987) Ann. N.Y. Acad. Sci. 512,97-114. Lacaze-Masmonteil, T., de Keyzer, Y., Luton, J.-P., Kahn, A. and Bertagna, X. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7261-7265. Layton, J.E., Scanlon, D.B., Soveny, C. and Morstyn, G. (1988) Cancer Res. 48, 4783-4789. Lehy, T., Accary, J.P.. Labeille, D. and Dubrasquet, M. (1983) Gastroenterology 84, 914-919. List, A.F., ~~nsworth, J.D., Davis, B.W., Hande, K.R., Greco, A. and Johnson, D.H. (1986) J. Clin. Oncol. 4, 1191-1198. Luster, W., Gropp, C., Sostmann, H., Kalbfleisch, H. and Havemann, K. (1982) Eur. J. Cancer Clin. Oncol. 18, 1275-1283. Luster, W., Gropp, C., Kern, H.F., Wahl, R., Roher, H.D. and Havemann, K. (1985) Recent Results Cancer Res. 99, 117129. MacIntyre, I., Alevizaki, M., Bevis, P.J.R. and Zaidi, M. (1987) Clin. Orthop. Relat. Res. 217, 45-55. McDonald, T.J., Nilsson, G., Vagne, M., Ghatei, M., Bloom, S.R. and Mutt, V. (1978) Gut 19, 767-774. Moody, T.W., Pert, C.B., Rivier, J. and Brown, M.R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75. 5372-5376. Moody, T.W., Pert, C.B., Gazdar, A.F.. Camey, D.N. and Minna, J.D. (1981) Science 214, 1246-1248. Moody, T.W., Bertness, V. and Camey, D.N. (1983) Peptides 4. 683-686.

12

Moody, T.W., Carney, D.N., Cuttitta, F., Quattrocchi, K. ayd Minna, J.D, (1985) Life Sci. 37, 105-113. Nakagawa, T., Mabry, M., De Bus&us, A., Ii&, J.N,, Nelkin, B.D. and Baylin, S.B. (1987) Proc. Natl. Acad. Sci. W.SA. 84, 5x3-5927. Naylor, St., Johnson, B.E., Mixma, J.D. and Sakaguchi, A.Y. (1987) Nature 329, 451-453. North, W.G., Maurer, H., V&in, H. and O’Donell, J.F. (1980) J. Ciin. Endocrinol. Metab. 51, 892-897. Noseda, A., Peeters, T.L., Delhayrc, M., Barmans, V,, Couvreur, Y., V~de~te~~ G., De ~r~quen, P., Roemans, P. and Yemauit, J.C. (1987) Thorax 42,7$4-789. Oates, E. and Herbert, E. (1984) 3. Biol. Chem. 259,7421-742.X Odell, W.D., Wolfsen, A.R., Bacbeiot, I. and Hirose, F.M. (1979) Am. J. Med. 66,631-638. OIson, N., Twiggs, L. and Sibley, R. (1982) Cancer 50,760-765. Pappas. T., Hamel, D., Debas, H., Walsh, J. and Tache, Y. (1985) Feptides 6,1001-IOUJ, Pearse, A.G.E. and Poll&, J.M. (1971) Gut 12, 783-788. Pert, C.B. and Schumacher, U.K. (1982) Lancet ii, 509-512. Pettengill, O.S., Wurster-Hiif, D.N., Gate, C.G. and Sorenson, G.D. (X9$5) Recent Results Cancer Rea 99,138-142. Pfluger, K.H., Koeppler, H., Havemann, K. and HoEe, R. {19SS) Eur. J. Haematol. 40,442~448. Pintax, J.E.: Schachter, B.S., Herman, A.B., Durgerian, S. and Krieger, D.T. (X984) Science 225,632-634. Pittman, S., Russ& P.J., Jelbart, M., Wass, J. and Raghavan, D. (1987) Cancer Genet. Cytogenet. 26,165-169. ~rou~-~~rl~nd, L., Meunier, H., Cat@,J., Dumont, D., Gagne, B. and La&tie, F. (1983) J. Steroid B&hem. 19, 439-445. Ratcliffe, J.G., Podmore, J., Stack, B.H.R., Spilg, W.G.S. and Gropp, C. (1982) Br. 5. Cancer 45,230-238. Roth R.A. and Barchas, J.D. (1986) Cancer 57, 769-773. Sausville, E.A., Lebacq-Verheyden, A.-M., Spindd, E.R., Cuttitta, F., Gazdar, A.F. and Battey, J.F. (1986) J. Biol. Chem. 262, 2451-2457. Sayler, W.R., Sayier. D.C. and Eg&ston, J.G. (1976) Cancer 37, 958-973. Shows, T.R., J~aguj~~, A.Y. and Naylor, S.L. (1982) Adv. Human Genet. 12. 341-451. Silverman, A.L., Park, J.-G., Hamilton, S.R., Gazdar, A.F,, Luk, G.D. and BayIin, S.B. (1989) Cancer Res. 49, 34683473. Smith, A.I. and Funder, J.W. (1988) Endoer. Rev, 9, 159-179. Sorenson, G.D., Petten@, OS., Brinek~Johnsen. T., Gate, CC. and Maurer, L.H. {1981) Cancer 47, 1289-1296. Sorenson, CD., Gate, CC. and Pettengill, OS. (1985) Recent Results Cancer Res. 99, 143-157. Spindel, E.R., Chin, W.W., Price, J,, Rees, LX, Besser, G.M. and Habener, J.F. (1984) Proc. NatJ. Acad. Sci. U.S.A. 81, 5699-5703. Spindel. E.R., ZiIberberg, M.D., Habnez, J.F. and Chin, W.W. (1986) Proc. NatI. Acad. Sci. U.S.A. 83,19-23.

Springall, D.R., fbrahim, N., Rode, J., Sharp, M., Bloom, S.R. and Pdak, J.M. (1986) J. Pathoi. lS0, 151-261. Steenbergh, P.H., Hoppener, J.W.M., Zandberg, J., Roes, B.A., Jansz, B.S. and Lips, C.J.M. (1984) 3. Endrocrinol. 67, 143-175. Strott, CA., Nugent, CA, and Tyler, F.H. (1968) Am. J. Med. 44,97-104. Suda, T,, Kondo, M., Totani, R., Hashimoto$ N., Suzuki, M., Imakai, T., Oda, T., Tomori, N., Yajima, F,, Sumitomo, T., Nakagami, Y., Usbiyama, T., Demura, K. and Sbizume, K. (1986) J. Clin. ~nd~~~o~. Metab. 63, 1047-1051. Taylor, J.E., Bogden, A.E., Moreau, J.-P. and Coy, D.H. (1988a) B&&em. Biopbys. Res. Commmt. 1X3,81-86. Taylor, J.E., Coy, D.H. and Moreau, J.-P. (1988b) Life Sci. 43, 421-427. Tsukada, T., Nakai, Y., Jingami, H., imura, H., Taii, S., Nakanisbi, S. and Numa, S. (1981) B&hem. Biophys. Res. Commun. 98, 535-540. Walsh, J.H., Wang, H.C, and Dockray, G.J. (1978) Fed. Pnx. 38,2215-2319. Weber, S,, Zuckerman, J.E., Bostwick, D.G., Bensch, KG,, Sikic, B.L. and Raffin, T.A. (1985) J. Clin. Invest. 75, 306-309. Wenk, RX., Bhagavart, B.S., Levy, R., Miller, D. and Weisburger, W. (1977) Cancer 40, 773-778. Wharton, J., P&k, J-M., Bloom, S.R., Chat& M.A., Sol&a, Es, Brown, M.R. and Pearse, AGE. (1978) Nature 273, 76% 770. Wall, P.J., Coy, D.H. and Rozertgurt, E. (1988) B&hem.. Biophys. Res. Commun. 155, 359-365. Wood, SM., Wood, J.R., Gbatei, M.A., Lee, Y-C., O’Shaughnessy, D. and Bloom, S.R. (l981) J. C&t+ Endocrinol, Metab. 53, 1310-1322. Yamaguchi, R., Abe, K., Kameya, T., Ada&, X., Taguchi, S., Otsubo, K. and Yanaihara, N. (1983) Cancer Res. 43, 3932-3939. Yamap&i, K., Abe, IL, Adach, I., Kimura, S,, Suzuki, S., Sktimada, A., Kodama, T., Kameya, T. and Shimosato, Y. (1985) Recent Results Cancer Res. 99,107-116. Y~a8uc~, K., Kaoru, A.B.E., Ada&i, I., Otsubo, K., Hagasaki, K., Suznki, K., Manmo, K., Asanuma, F,, ~suG~~as~, T., Miyake, Y., Yosbinaga, K., Matsubara, S., Kodama, T., Rameya, T. and Sbimosato, Y. (1986) Jpa. J. Clin. Oncol. 16, 235-241. Zachary, I. and Rozengurt, E. (1985) Proc. Nati. Acad, Sci. U.S.A. 82, X16-7620. Zaidi, M,, Breimer, L.H. and Macfntyre, I. (1987) hp. Pbysid. 72, 371-408. Zeytin, F.N., Rusk, S, and L.eff, SE. (198’7) Endocrinology 121, 361-370.