WO1990003181A2

WO1990003181A2 - Peptide antagonists for use in cancer therapy, and the use of mas oncogene and its product

Info

Publication number: WO1990003181A2
Application number: PCT/GB1989/001121
Authority: WO
Inventors: Michael Robert Hanley; Michel Goedert
Original assignee: Medical Research Council
Priority date: 1988-09-24
Filing date: 1989-09-22
Publication date: 1990-04-05
Also published as: WO1990003181A3; GB8822483D0; AU4324089A

Abstract

It has been demonstrated that the mas oncogene encodes an angiotensin receptor. A substance which blocks the biological activity of angiotensin may thus be used in the treatment or prevention of tumour development or ectopic hormone production. Suitable substances include angiotensin antagonists, partial agonists to angiotensins, and antibodies to angiotensins. The use of the product of the mas oncogene in an assay system for angiotensin-blocking activity is also claimed.

Description

PEPTIDE ANTAGONISTS FOR USE IN CANCER THERAPY, AND THE USE OF MASONCOGENE AND ITS PRODUCT

Field of the invention

This invention relates to developments concerning the mas oncogene.

Background to the invention

The mas oncogene was first identified and reported in 1986 (reference 1) by a group in Cold Spring Harbour, but has been studied further in only one other referecd paper (reference 2). This gene is a normal human gene which has transforming and tumour-inducing activity in standard assays, and its structure has been completely determined. The gene is freely available.

Summary of the invention

The present invention is based on work which demonstrates that the mas oncogene encoded an angiotensin receptor .

In particular, the inventors have introduced the mas gene into the NG115-401L neuronal cell line, and established that it was transcriptionally active by Northern blots using full-length clones and short cloned fragments d.s probes. The hypothesis was proposed that the mas oncogene would correspond to a peptide receptor regulating the inositol lipid/calcium signalling pathways, and stimulating target cell growth (reference 3), based on its very close sequence resemblance to an identified peptide receptor, the substance K receptor.

In screening a variety of cells which were expressing the foreign mas gene, the inventors have established that, in all cases, they had acquired sensitivity to the circulating hormone angiotensin II. They have assayed this using the fluorescent indicε tor Fura-2, which records changes in cellular calcium, and have shown by this means that the transfected cells express a receptor responding to the naturally-occurring angiotensins in the rank order angiotensin III greater than or equal to angiotensin II much greater than angiotensin I, when precautions were taken to exclude differential degradation by surface peptidases. The response was blocked by specific angiotensin antagonists, and not by bradykinin antagonists.

Independent assays, including activation of high affinity GTPase and production of inositol phosphates, have confirmed that the transfected cells have received functional angiotensin receptors. Significantly, the transfected cells respond to angiotensin III by a stimulation of DNA synthesis and growth, whereas they do not to the related peptide hormone bradykinin, whose responses in this cell line have been characterised in detail. This indicates that the mas oncogene can induce cells to grow in hormone-regulated manner, as would be predicted.

This information has been used to evaluate a number of tumour-derived cell lines and normal tissues for the expression of the mas transcript. The mas transcript has a one-to-one correspondence with cells and tissues known to be sensitive to angiotensins. It is particularly frequent in neuroblastoma or pheochromocytoma-de ived cell lines. In normal tissues, it is found in kidney vascular sites (including heart), and regions in brain, where it is enriched in cerebral cortex. This distribution pattern has been examined in humans and non-human species, and broad similarities found.

The implications may be that mas is a potential target for the initiation of human tumours. This is supported by the recognition that the humas mas gene maps to the distal end of the long arm of chromosome 6; a region known to have identified fragile breakpoints associated with neuroblastoma and pheochro ocytoma tumours. Thus, angiotensin antagonists and other substances which block the biological activity of angiotensin may have a novel and heretofore unsuspected activity as anti-neoplastics or anti-tumour drugs.

There is also evidence that the expression and activation of mas may be linked to the induction of other hormone genes in the transfected cell line. In a variety of neuroendocrine tumours, it is frequently observed that they produce a complex variety of physiologically inappropriate peptide and non-peptide hormones. This phenomenon, termed "ectopic hormone production", has not been understood, but may be related to the activation of mas and related oncogenes, suggesting that angiotensin antagonists may have therapeutic application in reversing some instances of ectopic hormone production.

Hence, in one aspect the present invention concerns use of a substance which blocks the biological activity of angiotensin in the treatment or prevention of tumour development or ectopic hormone production. - A -

Suitable substances include angiotensin antagonists (of which several are known, e.g. as potential drugs for alleviation of high' blood pressure), partial agonists to angiotensins, and antibodies to angiotensins.

Known angiotensin antagonists (some of which may act as partial agonists) include the following:

1 8

Sarcosine , Alanine -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Ala)

Sarcosine 1, Glycine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Gly)

Sarcosine 1 , Isoleucine 8 -antiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, lie)

1 8

Sarcosine , Leucine -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Leu)

Sarcosine 1, Valine5, Alanine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, Val, His, Pro, Ala)

Q

Isoleucine -angiotensin III (claimed to be specific for AIII)

(i.e. Arg, Val, Tyr, lie. His, Pro, Phe, lie)

The broad spectrum peptide antagonist D-Arg 1, D-Pro2' D-

7 9 -j x

Trp ' , Leu -substance P (i.e. D-Arg, D-Pro, Lys, Pro,

Gin, Gin, D-Trp, Phe, D-Trp, Leu, Leu) is also useful in this connection.

The invention is thought to be particularly applicable to neuroendocrine and neuroblastoma tumours, for preventing, arresting or retarding tumour growth and possibly even reversing tumour growth.

In a further aspect the present invention provides a method of treating or preventing tumour growth or ectopic hormone production, comprising administering a substance which blocks the biological activity of angiotensin.

The invention also concerns use of a substance which blocks the biological activity of angiotensin for preparation of a medicament for use in prevention or treatment of tumours or ectopic hormone production.

The results also suggest that antibodies, particularly monoclonal antibodies, to mas may be novel diagnostic reagents for tumour classification and potentially for targeting and therapy by immunotoxins .

Hence in another aspect the invention concerns use of antibodies to the mas oncogene in tumour diagnosis and for targeting of drugs in treatment of tumours and ectopic hormone production.

Antibodies to the m s oncogene may be produced in conventional manner, using well known techniques.

The invention also includes within its scope a cell line transfected with the mas oncogene.

Tn yet a further aspect the invention provides an assay system for substances which block the biological activity of angiotensin, using the product of the mas oncogene, e.g. obtained from a cell line trans fected with the mas oncogene. In this way further substances of potential use in the treatment or prevention of tumour development or ectopic hormone production may be identified.

The following description concerns work which demonstrates that the mas oncogene encodes an angiotensin receptor and refers to the accompanying drawings in which:

Figure 1 is a series of graphs illustrating angiotensin responses in Xenopus oocytes injected with synthetic mas RNA;

Figure 2A illustrates mtracellular Ca 2+ transients in parental NG115-401L or transfected 401L-C3 cell populations in response to angiotensin II (A II, lute) and bradykinin (BK, 2uM) ;

Figure 2B illustrates dose-dependence of peak intracellular calcium concentration [Ca 2+] . responses for angiotensin I, II or III in 401L-C3 cell populations;

Figure 3A illustrates intracellular Ca 2+ transients in response to angiotensin II (All, lOuM) and bradykinin

(BK,2uM) in 401L-CC3 cell populations;

Figure 3B illustrates the effect of acute TPA (200nM)

2+"| treatment on [Ca . responses to angiotensin II (All, luM) and bradykinin (BK, 200nM) in 401L-C3 cell populations;

Figures 4A and B are Northern blot analyses of poly (A)

RNA from parental NG11._-401L (lane 1) and trans fected 401L-C3 (lane 2) using P labelled human mas oncogene DMA (Fig 4A) or mouse beta-actin DNA (Fig 4B) as a probe. The blot was hybridized with the actin probe following dehybridization of the mas probe; and

Figure 4C is a Northern blot analysis of poly (A) RNA

32 from control human brain using P-labelled human mas oncogene as a probe with an autoradiographic exposure time of 5 days: lane 1, frontal cortex; lane 2, hippocampus; lane 3, cerebellum.

Description

Referring to Figure 1, angiotensin responses in Xenopus oocytes injected with synthetic mas RNA were determined as follows .

A 1.3 kilobase (kb) Bam Hl/Nsi I fragment containing the entire coding region of the human mas oncogene (reference

1) was subcloned into pGEM-7ZF (+). The plasmid was linearized by digestion with Nsi I and transcribed in vitro (reference 4) using T7 polymerase. Transcripts were η capped with m G(5 ' )ppp(5 ' )G (reference 5). The synthetic RNA gave a single band of about 1.3kb on a 1.5% formaldehyde gel. RNA was injected into defolliculated Xenopus oocytes at a concentration of lng.nl (volume injected per oocyte, about 50nl ) and incubated at 17°C for 1.5-3 days in Barth's medium supplemented with 2.5mM pyruvate. Physiological recordings were made at 20-23 °C using standard two-electrode voltage clamp techniques. Oocytes were continuously perfused with frog Ringer saline (about 2 ml min ; volume of recording chamber, 0.5ml). Drugs were bath applied for 45 sec and oocytes were rinsed for 5-20 min between drug applications. Uninjected oocytes showed no response to lOOnM angiotensin I, II or III. As shown in Figure IA, nanomolar concentrations of angiotensin III evoked dose-dependent currents. The bars above the traces indicate duration of drug applications. Calibration bars in Figure 1 are as follows: vertical, lOOnA (A.), 50nA (B.,C); horizontal 1 min.

Figure IB illustrates the relat.ive potency of angiotensins I, II and III. The responses to lOOnM Al (left) were significantly smaller than those induced by lOOnM All or AIII (centre and right). Recordings are from a single oocyte, different from the one shown in part A.

Figure 1C illustrates the effects of peptide receptor antagonists tested in an oocyte injected 3 days earlier with mas RNA: left, current response induced by lOnM AIII;

1 2 7 9 11 centre, application of lOuM [D-Arg ,D-Pro ,D-Trp ,Leu ]

- substance P (i.e. D-Arg, D-Pro, Lys, Pro, Gin, Gin, D-

Trp, Phe, D-Trp, Leu, Leu) (open bar ) reversibly inhibited the response to lOnM AI II ( solid bar ) ; left, the putative angiotens in II receptor antagonist ( Sar 1 , Val5 , Ala 8 ) -Al l

( i . e . Sar , Arg, Val, Tyr, Val , His, Pro, Ala ) (open bar , lOuM) weakly activated the receptor.

Thus, Injected oocytes expressed a novel sensitivity not observed in uninjected oocytes. Under voltage-clamp conditions, oocytes injected with mas-RNA exhibited a dose-dependent induction of an inward current in response to bath applied angiotensins I (Al ) , II(All) , and III(AIII) (Figure A, n=16 oocytes). No response was observed to bath application of bradykinin (lOOnM) or to a variety of other peptides. The rank order of potency of mammalian antiotensins was AIII greater than or equal to All much greater than Al (Figure IB). The responses were reversibly blocked by the broad spectrum peptide

1 2 7 9 11 antagonist, (D-Arg , D-Pro , D-Trp ' , eu ) -substance P

(reference 18) (Figure 1C), but not by the suggested angiotensin antagonists (reference 19), (Sar 1, Val5,

Ala )-AII and (Sar¹,Val )-AII (i.e. Sar, Arg, Val, Tyr,

Val, His, Pro, Phe), both of which show weak agonist activity (see Figure 1C) The angiotensin responses clearly displayed the delayed onset and oscillations characteristic of inositol trisphosphate-mediated calcium activation of endogenous chloride channels (reference 6).

This implies that the injected mas-RNA programmes the expression of angiotensin sensitive-receptors which couple into the endogenous inositol lipid/Ca -mobilising pathway. Similar coupling and functional responses have been obtained with expressed serotonin and substance K receptors (references 7 and 8).

To extend the functional analysis, a neural cell line NG115-401L was transfected with the mas gene; responses to peptide receptors working through the inositol lipid pathways have been characterised in detail in this system (reference 9), which does not express detectable levels of the mas transcript (see Figure 4A) .

Sma I linearized pMS422 (reference 1) and Pst I linearized pSV2neo were co-transfected into NG115-401L cells by calcium phosphate precipitation (reference 10). Cells were grown to confluence in the presence of G418 (1.8 mg/ml, Gibσo) to select for trans fectants, and pooled

G418 cell populations were screened for angiotensin sensitivity by (Ca 2+) . responses using the intracellular calcium indicator, Fura-2 (reference 11). These cells were then cloned by limiting dilution, and a number o£ individual angiotensin-responsi e clones were obtained. For [Ca2+] determinations, cells were grown on coverslips and loaded with Fura-2/AM; [Ca 2+]. was monitored by Fura-2 fluorescence at 500nm with excitation at 340nm, and calibrated using digitonin and manganese chloride as described previously (reference 12). The results are shown in Figure 2. In Figure 2A traces are representative of at least 3 separate determinations, and in Figure 2B each data point represents eanis.e.m. for 3-9 separate determinations.

One of the angiotensin-responsive clones, 401L-C3, was selected for detailed characterisation. By Northern blot analysis, it expressed a 2.4kb transcript when probed with a 0.35kb mas probe (Figure 4A and 4B).

Considering the matter in more detail, a 0.35kb Kpn l/Sall fragment from the mas oncogene clone p S422 (reference I) was subcloned into M13mpl8 and single-stranded DNA probes prepared as described (reference 20). RNA extractions, hybridizations and washings were performed as described

(reference 20). Each lane contained 20ug poly (A) RNA. The autoradiographic exposure times were 6 days for Figure 4A and lh for Figure 4B. The 0.24-9.5 kb RNA ladder

(Bethesda Research Laboratories) served as a size marker in the blots of Figure 4.

Figure 2A shows the responses of transfected and parental populations. The dose-response profiles for cytosolic

[Ca 2+ ] . elevation in 401L-C3 cells ( Figure 2B) exhibi t the same rank order as found for oocyte current stimula tion; AI II greater than or equa l to Al l much greater than Al , sugges ting that this pharmacology is an i ntrins ic feature of the receptor . In Figure 3A the pattern of cytosolic calcium changes is compared to those induced by bradykinin, using methods as described in connection with Figure 2. It can be seen that although the maximal response to angiotensin II is similar to bradykinin, it shows slower kinetics (about 5s and 15s to peak, respectively). Pre-treatment of the cells with bradykinin can abolish the response to an iotensins, but pre-treatment of the cells with antiotensins attenuates, although does not abolish, the response to bradykinin. Like the oocyte response, angiotensin stimulation of an intracellular calcium transient can be blocked by the previously-described peptide receptor antagonist, unlike the bradykinin-induced transient. These two responses can also be distinguished by their sensitivity to acute phorbol diester treatment. Figure 3B indicates that acute treatment with TPA (12-0- tetradecanoylphorbol-13-acetate) can attenuate the response evoked by All, but is without effect on that evoked by bradykinin. This result indicates that protein kinase C may act directly on either the mas gene product or a transduction component, such as a G-protein, which is used by this receptor and not by the bradykinin receptor. These conclusions about the intracellular calcium transients elicited by angiotensins have been reproduced and extended using single-cell imaging approaches.

As the mas oncogene has been shown to be tumou igenic in nude mice, and has transforming activity on NIH-3T3 cells (reference 1), the ability of angiotensins to stimulate DNA synthesis in the 401L-C3 clone was examined. As the Table shows, angiotensins stimulate [ H]-thymidine incorporation into serum-starved cells, whereas bradykinin does not . The tissue distribution of the mas proto-oncogene transcript in normal human brain and in peripheral tissues has also been examined using the 0.35 kb mas probe and techniques as described above in connection with Figure 4. The human tissues used were obtained less than 3h after death. As shown in Figure 4C, the highest level of the transcript was found in human cortex, with lower levels in hippocampus and cerebellum, similar to the results found for rat brain (reference 13). The mas transcript was expressed at very low levels in human liver, kidney and adrenal glands. These results are consistent with the anticipated peripheral distribution of an angiotensin receptor. There may exist a multigene family whose protein products respond to angiotensins, and there may be one or more receptors for each of the naturally-occurring molecular forms of angiotensins (Al, All and AIII). Earlier observations (reference 14) reported that both brain angiotensin-binding sites and functional responses appeared to prefer AIII over All. Thus, the mas oncogene may encode a "neuronal-type" angiotensin receptor.

The identification of the mas proto-oncogene product as an angiotensin receptor with mitogenic activity strongly suggests that at least some neural peptides may function as growth factors (references 15 and 16) and thus could be important during the development of the nervous system. It also indicates that deregulation of the inositol lipid signalling pathways could, in some circumstances, be sufficient for tumourigenesis. It is interesting to note that the location of the human mas gene on chromosome 6 (reference 2) is within a region of fragile sites associated with neuroblastoma and other tumours. Little is known of the activation of the mas proto-oncogene, but earlier work (reference 1) suggested that the proto- oncogene itself had intrinsic tumorigenic capacity. Rearrangement of the 5' -flanking sequence confers enhanced tumorigenicity. Additionally the human mas oncogene shows only weak transforming activity in NIH-3T3 cells, but is paradoxically quite potent in eliciting tumours in- vivo. This result may now be explicable with the realisation that tumour induction may require stimulation of the mas gene product by its cognate ligand, an angiotensin. Accordingly, the actions of exogenously administered angiotensins or suitable antagonists in promoting or retarding mas-induced tumour formation in- vito should now be assessed. This is the first oncogene for which a defined pharmacology, including potential antagonists, is available.

These results are also interesting in a pathological context different from carcinogenesis. For some time, angiotensin dysfunction has been proposed as a possible mechanism in genetic and experimental hypertension (reference 17). Recent hypotheses on the origin of essential hypertension (reference 21) have emphasised the likely role of vascular hypertrophy following chronic exposure* to an endogenous growth factor. It is an appealing possibility that the endogenous growth factor- may be an angiotensin.

Table

[ H] thymidine incorporation in parental NG115-40 IL and transfected 401L-C3 cell o ulations

[ H]Thymidine incorporation into serum-starved parental

NG115-401L and transfected 401L-C3 cells. Cells were

4 plated at low density (less than or equal to 1 x 10 cells per cm 2) in 35-mm tissue culture dishes in DMEM containing

5% FCS; Ih later the medium was replaced with serum-free

DMEM, then after 24 h cells were stimulated as appropriate in the presence of 0.5uCi ml -1 [3H]thymidine (New England

Nuclear). After 48 h further incubation, the radioactivity incorporated into trichloroacetic acid- insoluble material was assessed by liquid scintillation counting (reference 18). Data are means +s.e.m. of at least three separate determinations each performed in triplicate. Radioactivity present in controls: NG115- 401L 15,469+_1,567 c.p.m.; 401L-C3 15,209+735 c.p.m. significance was determined by a two-tailed student's t- test.

* Different from control, P less than 0.06. **401L-C3 different from NG115-401L,p less than 0.05. ND,

SUBSTITUTESHEET References

1. Young, D., Waitchec, G., Birchmeier, C. , Fasano, G., and Wigler, M. Cell 45, 711-719 (1986).

2. Rabin, M., Birnbaum, D., Young, D., Birchmeier, C. , Wigler, M. , and Ruddle, F.H. Oncogene Res. 1, 169-178 (1988) .

3. Hanley, M.R., and Jackson, T.R. Nature 329, 766-767 (1987) .

4. Melton, D.A. et al. Nucleic acids Res. 12, 7035-7056 (1984).

5. White, M.M. et al. Proc. natn. Acad. Sci . U.S.A. 82, 4852-4856 (1985).

6. Gillo, B., Lass, Y., Nadler, E. and Oran, Y., J. Physiol. Lond. 392, 349-361 (1987).

7. Julius, D. e_t_ al. Science 241, 55.

8. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y. , Kuno, M. and Nakanishi, S. Nature 329, 836-838 (1987).

9. Jackson, T.R. , Hallam, T.J., Downes, C.P., and Hanley, M.R., EMBO J. 6, 49-54 (1987).

10. Wigler, ... Pellicer, A., Silverstein, S. , Axel, R. , Urlaub, G., and Chasin, L. Proc. natn. Acad. Sci . U.S.A. 76, 1373-1376 (1979). 11. Grynkiewicz, G., Poenie, M. , and Tsien, R.Y., J. biol. Chβ . 260, 3440-3450 (1985).

12. Jackson, T.R., Patterson, S.I., Thastrup, 0., and Hanley, .R. Biochem. J^ 253, 81-86 (1988).

13. Young, D., O'Niell, K., Jessell, T., and Wigler, M. , Proc. natn. Acad. Sci. U.S.A. 85, 5339-5342 (1988).

14. Burt, D.R., in Neurotransmitter Receptors, Part I. Series B, 9 (Eds. Enna, S. .. and Yamamura, H.I.) Chapman and Hall, London, (1980).

15. Hanley, M.R. , Nature 315, 14-15 (1985)

16. Zachary, I., et_ a3.. Devi Biol. 124, 295-308 (1987).

17. Ferrario, CM. Hypertension 5, V73-V79 (1983)

18. Corps, A.N., Rees, L.H., and Brown, K.D., Biochem. J. 231, 781-784 (1985).

19. Regoli, D., Can. J. Physiol. Pharmacol. 57, 129-139 (1979).

20. Goedert, M., EMBO J. 6, 3627-3632 (1987).

21. Connell, J.M.C. Trends pharmac. Sci. 7 , 412-418 (1986).

SUBSTITUTE SHEET

Claims

1. Use of a substance which blocks the biological activity of angiotensin for preparation of a medicament for use in the treatment or prevention of tumour development or ectopic hormone production.

2. A use according to claim 1, wherein the substance comprises one or more of the following: angiotensin antagonists, partial agonists to angiotesins, and antibodies to angiotensins.

3. A use according to claim 2, wherein the substance comprises one or more of the following:

Sarcosine 1, Alanine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Ala);

Sarcosine 1, Glycine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Gly) ;

Sarcosine 1, Isoleucine8 -antiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, lie);

Sarcosine , Leucine -angiotensin II

(i.e. Sar, Arg, Val, Tyr, .lie, His, Pro, Leu):

Sarcosine 1 , Vali.ne^'5, Alani.neR -angiotensin II

(i.e. Sar, Arg, Val, Tyr, Val, His, Pro, Ala); and Q

Isoleucine -angiotensin III

(i.e. Arg, Val, Tyr, lie, His, Pro, Phe, lie).

4. A use according to claim 2, wherein the substance comprises the broad spectrum peptide antagonist D-Arg , D- Pro 2, D-Trp7'9, Leu11-substance P (i.e. D-Arg, D-Pro,

Lys, Pro, Gin, Gin, D-Trp, Phe, D-Trp, Leu, Leu).

5. A method of treating or preventing tumour growth or ectopic hormone production, comprising administering a substance which blocks the biological activity of angiotensin.

6. A method according to claim 5, wherein the substance comprises one or more of the following: angiotensin antagonists, partial agonists to angiotensins, and antibodies to angiotensins.

7. A method according to claim 6, wherein the substance comprises one or more of the following:

Sarcosine 1, Alanine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Ala);

Sarcosine 1, Glycine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, lie, His, Pro, Gly) ;

1 8

Sarcosine , Isoleucine -antiotensin II

(i.e. Sar, Arg, Val, Tyr, He, His, Pro,. lie);

Sarcosine 1 , Leucine8 -angiotensin II

(i.e. Sar, Arg, Val, Tyr, He, His, Pro, Leu): Sarcosine , Valine , Alanine -angiotensin II (i.e. Sar, Arg, Val, Tyr, Val, His, Pro, Ala); and g

Isoleucine -angiotensin III

(i.e. Arg, Val, Tyr, He, His, Pro, Phe, He)

8. A method according to claim 6, wherein the substance comprises the broad spectrum peptide antagonist n-Arg , D- V 9 ^~ 1 Pro , D-Trp ' , Leu -substance P (i.e. D-Arg, D-Pro, Lys,

Pro, Gin, Gin, D-Trp, Phe, D-Trp, Leu, Leu).

9. A method according to claim 5, for treatment of neuroendocrine and neuroblastoma tumurs, or for preventing, arresting, retarding or reversing tumour growth.

10. A cell line transfected with the mas oncogene.

11. An assay system for substances which block the biological activity of angiotensin, using the product of the mas oncogene .