Radioimmunoassays for the C-terminus of prothymosin α and the N-terminus of parathymosin α for the measurement of the levels of α-thymosins in human cancer

JOURNALOF IMMUNOLOGICAL METHOOS ELSEVIER

Journal of Immunological Methods 169 (1994) 163-171

Radioimmunoassays for the C-terminus of prothymosin a and the N-terminus of parathymosin a for the measurement of the levels of a-thymosins in human cancer O . E . T s i t s i l o n i a, E. H e i m e r b A . Felix h p . p . Y i a l o u r i s a, j. V a m v o u k a k i s c W . V o e l t e r d ) A . A . H a r i t o s *'a a Zoological Laboratory, Faculty of Science, University ofAthens, GR 15784, Athens, Greece, b Peptide Research Department, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA, c Health Department, Hellenic Telecommunications Organization, 99 Kifissias Avenue, GR 151 24, Athens, Greece, a Abteilung fiir Physikalische Biochemie, Physiologish-chemisches lnstitut der Universitiit T'dbingen, Hoppe-Seyler Str. 4, D-7400 Tiibingen, Germany

(Received 29 April 1993, revised received 28 September 1993, accepted 4 October 1993)

Abstract A radioimmunoassay specific for the C-terminus of human prothymosin a was developed using the synthetic peptide [Cys-Aca°]-human prothymosin a (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca°]-hu man prothymosin ~ (90-109)-OH labelled with 1251 as tracer. The radioimmunoassay measured intact prothymosin a, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin a. A major epitope was located in the segment 95-107. A radioimmunoassay specific for the N-terminus of human parathymosin a , also measuring intact parathymosin a in the range of 1-20 pmol and not cross-reacting with prothymosin a, was developed using the synthetic peptide [Cys-Aca°]-human parathymosin a (1-30)-OH as antigen coupled to KLH and the analogue [Tyr-Aca°]-human parathymosin a (1-10)-OH labelled with 1251 as tracer. A major epitope was located in the segment 1-10. These radioimmunoassays, together with a previously established radioimmunoassay for the N-terminus of prothymosin a, permitted the identification of the molecular forms of the cross-reactive materials in both normal and neoplastic breast tissue extracts as intact prothymosin a and parathymosin a. It was also possible to reveal significantly higher levels of both a-thymosins in breast cancer tissue compared to the nearby healthy tissue - the mean of 14 samples was over 14-fold higher - suggesting a role of both prothymosin a and parathymosin a in cell proliferation. The reported radioimmunoassays are expected to facilitate the search for prognostic a n d / o r diagnostic applications of these polypeptides in human cancer. Key words: Prothymosin a; Parathymosin a; Thymosin al; Cancer; Epitope; Radioimmunoassay

1. Introduction H u m a n p r o t h y m o s i n a (109 r e s i d u e s ) a n d p a r a t h y m o s i n a (101 residues), t h e e n d o g e n o u s * Corresponding author.

m e m b e r s o f t h e a - t h y m o s i n family, have b e e n c h a r a c t e r i z e d for t h e i r p r i m a r y s t r u c t u r e b o t h by Edman degradation and cDNA sequencing ( G o o d a l l et al., 1986; P a n et al., 1986; C l i n t o n et al., 1989). T h e y show s t r u c t u r a l similarity p a r t i c u larly at t h e i r N - t e r m i n i . T h y m o s i n a 1 has b e e n

0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(93)E0286-Q

164

O.E. Tsitsiloni et al. /Journal o f Immunological Methods 169 (1994) 163-171

shown to represent an N-terminal fragment (residues 1-28) of prothymosin a (Haritos et al., 1984a). a-thymosins are ubiquitous in mammalian tissues and show a differential tissue expression with thymus being the richest source of prothymosin a and liver of parathymosin a (Haritos et al., 1984b). The levels of both athymosins diminish with age in human tissues with the sharpest decrease for prothymosin a in thymus in the first 10 years of life (Tsitsiloni et al., 1993). The physiological role of a-thymosins is unclear. For prothymosin a an intracellular role related to cell growth (Eschenfeldt and Berger, 1986; Conteas et al., 1990; Eilers et al., 1991; Sburlati et al., 1991; Tsitsiloni et al., 1993) and an extracellular role related to the enhancement of cell-mediated immunity phenomena (Baxevanis et al., 1987; Baxevanis et al., 1988; Baxevanis et al., 1992) have been suggested. The development of specific radioimmunoassays for the C-terminus of prothymosin a and the N-terminus of parathymosin a has already permitted the investigation of developmental changes of the levels of a-thymosins in healthy human tissues and revealed the relationship of increased levels of these polypeptides in solid tumours in man (Tsitsiloni et al., 1993). These radioimmunoassays are expected to assist in understanding the relationship of a-thymosins in cancer with a potential for improved diagnosis. Moreover they are expected to facilitate the search for the physiological role of these essential polypeptides by permitting the concomitant detection and measurement of the levels of the polypeptides in human tissue extracts and body fluids, in both health and disease.

2. Materials and methods

2.1. Extraction of a-thymosins from human breast tissues Samples of healthy human breast tissue and neighbouring malignant growth were obtained during surgery and stored immediately at -45°C. The samples were provided by Drs. J. Gogas and

Ch. Markopoulos (Laikon Hospital, St. Thomas Square, Goudi, in Athens). The frozen tissues were pulverized on dry ice using a mortar and pestle. The powder was added in approximately 10 vols. of boiling water and boiling continued for 5 more min. The suspension was stored at 4°C overnight, homogenized the next day in a Sorval blender homogenizer with three 45 s bursts at full speed. To the homogenate 0.1 vol. of 10 M formic acid/2 M pyridine buffer, pH 2.9, was added. The sample was stored at - 10°C overnight. After thawing the sample was centrifuged at 12,000 x g in a Sorval RC2B centrifuge and the supernatant was passed through a funnel filled with glass wool. The sample was concentrated in a SpeedVac centrifugal concentrator (Savant) before being subjected to HPLC gel filtration. 2.2. Peptide synthesis and conjugation to carrier protein The synthesis of the peptides human prothymosin a (90-109)-OH, [Cys-Aca°]-human prothymosin a (90-109)-OH, [Tyr-Aca°]-human prothymosin a (90-109)-OH, human parathymosin a (1-30)-OH, [Cys-Aca°]-human parathymosin a (1-30)-OH and [Tyr-Aca°]-human parathymosin a (1-10)-OH was performed using the Merrifield solid-phase methodology (Barany and Merrifield, 1982). Aca stands for aminocaproic acid. After cleaving from the resin, the crude peptides were purified to homogeneity by HPLC and gave the expected amino acid composition after acid hydrolysis. [Cys-Aca°]-human prothymosin a (90109)-OH and [Cys-Aca°]-human parathymosin a (1-30)-OH were both coupled to an equal weight of the carrier protein, keyhole limpet hemocyanin (KLH), using m-maleimidobenzoyl-N-hydroxysuccinimide as the linking reagent. The conjugation and subsequent workup was carried out as described (Green et al., 1982).

2.3. Radioimmunoassay for the N-terminus of prothymosin a This was performed as described by Yialouris et al. (1988).

O.E. Tsitsiloniet al. ~Journal of Immunological Methods 169 (1994) 163-171 2.4. Radiolabelling of tracers The tracers [Tyr-Aca°]-human prothymosin a (90-109)-OH and [Tyr-Aca°]-human parathymosin a (1-10)-OH were radioiodinated by the chloramine T method as previously reported (Tsitsiloni et al., 1988); 1.25 mCi of ~25I (Amersham) was used to label 0.5 /zg of each synthetic peptide. 2.5. Other materials Prothymosin a was isolated from human thymus (Pan et al., 1986) and parathymosin a from rat liver (Komiyama et al., 1986). 2.6. Fragmentation of antigens Proteolytic digestions were carried out as following. (1) Staphylococcus aureus V8 protease: 140/zg of dry synthetic human prothymosin a (90-109)O H or human parathymosin a (1-30)-OH was dissolved in 100/zl of 0.1 M ammonium acetate, 2 mM E D T A p H 4.0, to which 5 / z g of the protease was added. The sample was incubated for 15 h at room temperature and subsequently lyophilized. (2) Thermolysin: 140/~g of dry synthetic human prothymosin a (90-109)-OH or human parathymosin a (1-30)-OH were dissolved in 2 5 / z l of 0.12 M ammonium bicarbonate p H 7.5, to which 10 /zg of thermolysin were added. The sample was incubated for 2 h at 37°C and subsequently lyophilized. (3) TPCK-trypsin: 140 ~g of dry synthetic human prothymosin a (90-109)-OH or parathymosin a (1-30)-OH were dissolved in 60/~1 of 0.4 M pyridine pH 7.0, to which 15 /~g of TPCKtrypsin, dissolved in 1 mM HC1, were added. The sample was incubated for 15 h at room temperature. The reaction was terminated by the addition of 4.6/zl of formic acid and the sample was subsequently lyophilized. The proteolytic fragments were isolated on a Merck Lichrospher 100 A °, 5/~, C18 RP-column with a gradient of 0 - 2 1 % acetonitrile in water (for fragments of the C-terminus of prothymosin

165

a ) or 0 - 3 3 % (for fragments of the N-terminus of parathymosin or) over 60 min. Both solvents contained 0.1% trifluoroacetic acid. Fragments were identified by amino acid composition analysis using the PicoTag method (Waters Ass.).

3. R e s u l t s

3.1. Radioimmunoassay for the C-terminus of prothymosin a Rabbits were immunized subcutaneously at monthly intervals with [Cys-Aca°]-human prothymosin a (90-109)-OH coupled to KLH. For each immunization 0.4 mg of the polypeptide was injected in the form of an emulsion (1:1) with complete Freund's adjuvant. [Tyr-Aca°]-human prothymosin a (90-109)-OH labelled with 1251 was employed as tracer. For the radioimmunoassay, 1.25 ~1 of the antiserum (designated Chr) and 30,000 cpm of tracer were used. This radioimmunoassay cross-reacted 100% with the intact polypeptide, human prothymosin a, on an equimolar basis (Fig. 1). The useful range was 2-100 pmol of prothymosin or. No

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Fig. 1. Comparison of radioimmunoassays for the C-terminus of prothymosin a. Prothyrnosin a, its C-terminal fragment prothymosin a (90-109)-OH and parathymosin a were separately incubated with the tracer [TyrlZSI-Aca°]-humanprothymosin a (90-109)-OH. Subsequent displacement of the tracer was assessed and any cross-reactivityindicated by calculating the percentage of tracer remaining bound to the antiserum.

166

O.E. Tsitsiloni et a!/Journal of Immunological Methods 169 (1994) 163-171

100 ,*t'E

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right compared to that of the antigen (segment 90-109). Segment 95-107 shares only two identical residues with the C-terminus of human parathymosin a (Fig. 3). Lesser cross-reactivities were also observed for the N- and C-termini of the antigen (i.e., segments 90-102 and 103-109) (Fig. 2).

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Rabbits were immunized subcutaneously at monthly intervals with [Cys-Aca°]-human parathymosin a (1-30)-OH coupled to KLH. For each immunization, 0.5 mg of the polypeptide was injected in the form of an emulsion (1:1) with complete Freund's adjuvant. For the tracer, [Tyr-Aca°]-human parathymosin a (1-10)-OH labelled with z25I was employed. For the radioimmunoassay 2 /xl of the antiserum (designated Vin) and 25,000 cpm of tracer were used. The displacement curve for parathymosin a was shifted to the right compared to that of the antigen showing an approximately two-fold lower cross-reactivity with the intact parathymosin a compared to the N-terminal segment (Fig. 4). No cross-reactivity was found with human prothymosin a (Fig. 4). The useful range of this radioimmunoassay was 1-20 pmol for rat parathy-

pmoles

Fig. 2. The ability of proteolytic cleavage fragments of the C-terminal sequence 90-109 of prothymosin a to compete for binding of the [Tyr125I-Aca°-human prothymosin a (90-109)OH derivative to the prothymosin a (90-109)-OH antiserum. Proteolytic cleavage products of prothymosin a (90-I09)-OH (see materials and methods section and Fig. 3) and prothymosin a (90-109)-OH were incubated with the tracer over the range of concentrations indicated. Subsequently, displacement of the iodinated tracer was assessed and any cross-reactivity indicated by calculating the percentage remaining bound to the antiserum.

cross-reactivity was observed with rat parathymosin a up to 100 pmol (i.e., approximately 1 /zg) (Fig. 1). The major epitope appears to be located within the 13 amino acid residue segment 95-107 (Fig. 2), since the displacement curve of this polypeptide was only slightly shifted to the

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peptides derived by cleavage with thermolysin (Th), S. aureus V8 protease (S), trypsin (T) and S. aureus V8 protease digestion followed by trypsin digestion (S/T). Sequence identity to the C-terminus of human parathymosin a and the N-terminus of human prothymosin or, respectively, are indicated by shaded boxes.

O . E . Tsitsiloni et aL / J o u r n a l

of Immunological

mosin a (the N-terminal segments 1-20 of human and rat parathymosin a are identical). A major epitope was found to be located in the segment 1-10 (Fig. 5). Segments lacking the first three residues (residues 4-14) or the last four residues of this epitope (residues 1-6) did not cross-react (Fig. 5). However, segment 1-10 was approximately 30 times less effective than the antigen in displacing the ligand.

Methods 169 (1994) 163-171

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In H P L C gel filtration experiments of healthy (Fig. 6, Control) and neoplastic (Fig. 6, Cancer) h u m a n breast tissue extracts, prothymosin a Cterminal immunocross-reactive material eluted as a single p e a k corresponding to the elution position of isolated prothymosin a. Also the N-terminal prothymosin a cross-reactive material eluted mainly at the position of isolated prothymosin a. The coincidence of the elution volumes of the Cand C-terminal immunocross-reactive materials with that of isolated prothymosin ot suggests that the cross-reactive material is intact prothymosin a in both healthy and neoplastic breast tissue.

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In Fig. 7, the H P L C gel-filtration profile of parathymosin ot cross-reactive material in healthy (Fig, 7, Control) and neoplastic (Fig. 7, Cancer) female h u m a n breast tissue extracts showed a single immunocross-reactive p e a k which corresponded to the elution position of isolated rat parathymosin a. The coincidence of the elution volumes of parathymosin a immunocross-reactive material with that of intact parathymosin a suggests the presence of intact parathymosin a in both healthy and malignant breast tissue.

40

3.4. Levels of prothymosin a and parathymosin a in human breast cancer

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pmoles Fig. 4. Comparison of radioimmunoassaysfor the N-terminus of parathymosin t~. Parathymosin a, its N-terminal fragment 1-30 and prothymosin a were separately incubated with the tracer, [TyrZESI-Aca°l-human parathymosin a (1-10)-OH. Subsequent displacement of the tracer was assessed and any crossreactivityindicated by calculating the percentage of tracer remaining bound to the antiserum.

In Table 1, the levels of prothymosin a and parathymosin a in healthy h u m a n breast tissue (Control) and nearby neoplastic (Cancer) tissue of 14 patients are shown. The histological type of cancer in all cases was grade II. T h e levels of prothymosin ot in the malignant tissue extracts were 3.3-90.6-fold higher c o m p a r e d to the nearby healthy tissue extracts, with a m e a n value of 22.7

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Table 1 Levels of ct-thymosins in paired healthy and grade II neoplastic human breast tissues extracts

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O.E. Tsitsiloni et al. / Journal of Immunological Methods 169 (1994) 163-171 0.04

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Fig. 7. Separation of parathymosin a (1-30)-OH cross-reactive material in healthy (Control) and neoplstic (Cancer) breast tissue extracts by gel filtration HPLC. Elution volume of isolated rat parathymosin a is indicated (III). Aliquots of 200 /zl of fractions for both control and neoplastic samples were analysed by the radioimmunoassay specific for the N-terminus of parathymosin a. Details of the HPLC gel-filtration experiment are given in Fig. 6.

detection of both prothymosin 6 and its C-terminal fragment(s) in tissue extracts. Previously the radioimmunoassay specific for the N-terminus of the same polypeptide was found to detect both prothymosin 6 and its biologically active N-terminal fragment thymosin 61 (Haritos and Horecker, 1985) in tissue extracts (where the action of endogenous proteases was not inhibited during tissue extraction). However a combination of the radioimmunoassay specific for the N-terminus of prothymosin 6 (Yialouris et al., 1988) with that specific for the C-terminus of prothymosin 6, reported here, applied to gel-filtration fractions, represents a simple test for the presence of intact prothymosin 6 in tissue extracts. The radioimmunoassay for the N-terminus of parathymosin 6 was developed because the existing antibodies had been raised against a partially incorrect sequence of the N-terminus of this

169

polypeptide (residues 1-4, Tsitsiloni et al., 1988) thereby resulting in a reduced cross-reactivity with parathymosin a, or against the whole parathymosin a molecule isolated from rat (Panneerselyam et al., 1987a). As antigens for generating polyclonal antisera specific for a limited protein sequence, we used synthetic peptides corresponding to (i) the 20 residue long C-terminus of human prothymosin a (residues 90-109) and (ii) the 30 residue long N-terminus of human parathymosin a, both coupled to KLH through a spacer attached to the N-terminus to increase antigenicity. Neither antisera showed any crossreactivity with the other a-thymosin. The equal cross-reactivity of the antiserum specific for the C-terminus of prothymosin a with intact prothymosin a differs from the behaviour of the antiserum raised against the N-terminus of prothymosin a (sequence 1-28) and tested with intact prothymosin a (Haritos and Horecker, 1985) which shows only 20% cross-reactivity. This might be caused by differing steric hindrance from the rest of the prothymosin ot molecule depending on whether antibody is bound to the C-terminus or the N-terminus. For the radioimmunoassay specific for the N-terminus of parathymosin a a tracer (ten residues long) shorter than the antigen (30 residues long) was selected. This was done in order to generate antibodies which better recognized the antigen within the intact parathymosin a molecule and at the same time avoided the cross-reactivity with the N-terminus of prothymosin a since only three residues are identical in the first ten residues of the two thymosins (Fig. 3) while 11 residues are identical in residues 11-30. The same strategy was successful for the radioimmunoassay for the N-terminus of prothymosin a (Yialouris et al., 1988), i.e., the antigen being the prothymosin a sequence 1-28 and the tracer an analogue of the sequence 1-10. The radioimmunoassay for the C-terminus of prothymosin a appears to recognise an epitope within sequence 95-107 and the case of the radioimmunoassay for parathymosin a a major epitope is located within sequence 1-10. Since an N-terminal epitope within residues 1-10 of prothymosin a has been reported previously (Haritos

170

O.E. Tsitsiloni et al. / Journal o f Immunological Methods 169 (1994) 163-171

and Horecker, 1985), it appears that the Nterminus of parathymosin a and prothymosin a show similar immunogenicity on top of their 47% sequence similarity (in residues 1-30). The useful range of the radioimmunoassay for the C-terminus of prothymosin ot was 2-100 pmol which is similar to that previously reported for the N-terminus of prothymosin a (1-60 pmol (Yialouris et al., 1988)). For parathymosin a it was 1-20 pmol, i.e., shifted to a lower range as compared to previous reports of 4-500 pmol (Panneerselvam et al., 1987a) and 5-450 pmol (Tsitsiloni et al., 1988). Moreover our assay differs from the previous parathymosin a radioimmunoassays since it recognises almost equally well both the Nterminus and the intact parathymosin a. An important application of these radioimmunoassays is in studies of the relevance of prothymosin a and parathymosin a in human cancer. With the radioimmunoassays for the N- and Ctermini of prothymosin a and parathymosin a, the crossreactive materials in normal and neoplastic human breast tissue extracts were characterized as intact prothymosin a and the Nterminus of parathymosin a, respectively. For prothymosin a this is in agreement with previous reports for the tissues of various mammals including human thymus (Haritos et al., 1984a; Pan et al., 1986; Panneerselvam et al., 1988; Low et al., 1990; Tsitsiloni et al., 1993;), blood plasma (Panneerselvam et al., 1987b) and malignant and healthy tissue extracts of human colon (Tsitsiloni et al., 1993). In the case of parathymosin a it is also in agreement with observations on various human tissues (thymus, liver, spleen) and healthy and malignant colon extracts (Tsitsiloni et al., 1993). The significantly higher levels of both athymosins in human breast cancer compared to healthy breast tissue (Tsitsiloni et al., 1993 and Table 1 in this manuscript) and colon cancer (Tsitsiloni et al., 1993), suggest a relationship between the increased levels of these polypeptides and tumours' growth and therefore with cell proliferation. Among the questions which remain to be addressed are the following. (a) Do the increased levels of prothymosin a and parathymosin a rep-

resent a more general phenomenon which also applies to other common and rare types of human cancer? (b) Do the levels of a-thymosins in blood plasma reflect the increases observed in the solid tumours? (c) Are the molecular forms of these polypeptides intact or fragments? and (d) Is there a prognostic a n d / o r diagnostic potential for a-thymosins in cancer?

5. Acknowledgements This work was supported in part by grants to A.A.H. from the International Atomic Energy Agency (GRE-6921) and the Hellenic Telecommunications Organization.

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