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Clinical Chemistry 43: 59-63, 1997;
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(Clinical Chemistry. 1997;43:59-63.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Development of ELISA to estimate thymosin {alpha}1, the N terminus of prothymosin {alpha}, in human tumors

Lourdes Loidi, Anxo Vidal, Juan B. Zalvide, José L. Puente1, Francisco Reyes1 and Fernando Domíngueza

1 Departamento de Cirugía, Facultad de Medicina, Hospital Xeral de Galicia, Santiago de Compostela, Spain.
a Author for correspondence. Fax (34) (81) 574145; e-mail fsfedopu{at}usc.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We reported that tumor content of prothymosin {alpha} (ProT {alpha}) is a proliferation index of human breast tumors that might be used to identify patients at high risk for distant metastasis (Dominguez et al., Eur J Cancer 1993;29A:893–7). In that study ProT {alpha} concentrations were measured by a RIA; here we present an alternative nonisotopic assay that could be used in a standard clinical laboratory. Main features of the ELISA are: (a) A recombinant fusion protein glutathione S-transferase (GST)–human ProT {alpha} was used to coat the microtiter plates; (b) we used a polyclonal antiserum raised in rabbits that detects thymosin {alpha}1, the NH2-terminal fragment of ProT {alpha}; (c) it is as sensitive as the RIA; (d) it is faster than the RIA. ProT {alpha} concentrations in various human tumors (skin, esophagus, colorectal, and breast) as assessed by ELISA were comparable with, although twofold greater than, the values previously estimated by RIA.


Key Words: indexing terms: cancer • immunoassay • cell proliferation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prothymosin {alpha} (ProT {alpha}) is a small, highly acidic protein first isolated as the precursor of thymosin {alpha}1 (1).1 ProT {alpha} appears to be the endogenous peptide from which thymosin {alpha}1 is formed by proteolytic modification during the tissue extraction procedure (1)(2). Although an extracellular role for ProT {alpha} cannot be ignored, present evidence suggests that it acts at a nuclear level (3)(4)(5)(6).

Data gathered over the last few years strongly suggest that ProT {alpha} may well be related to normal cell proliferation, and the evidence is as follows: (a) ProT {alpha} mRNA was induced in serum-deprived fibroblast 3T3 cells when they were stimulated to proliferate (7)(8), and ProT {alpha} mRNA expression is also correlated to the proliferative activity of T cells and a small intestine-derived cell line (9)(10); (b) immunohistochemical studies have also shown that ProT {alpha} is expressed in proliferating but not quiescent cells in all tissues studied so far (3)(11)(12)(13); (c) ProT {alpha} mRNA antisense oligomers were shown to inhibit cell division in myeloma cells (14); (d) activation of the transcription of the protooncogene Myc led to a rapid increase in the transcription of the ProT {alpha} gene, even in the absence of protein synthesis (15); previously, we showed that ProT {alpha} expression is correlated with the proliferating activity of a rat pituitary tumor cell line (16). Moreover, in 71 patients with classic NOS invasive ductal carcinomas, ProT {alpha} concentrations as assayed by a RIA that detects thymosin {alpha}1 were significantly greater in tumor samples than in normal breast tissue. ProT {alpha} concentrations were correlated with: (a) the number of positive axillary lymph nodes and (b) the percentage of tumor cells in the S phase plus the G2/M phase as assessed by flow cytometry. Of special relevance was the finding that ProT {alpha} concentrations might be used to identify patients at high risk for distant metastasis (17). Because of the potential clinical use of ProT {alpha} determination in human tumors, we decided to develop a nonisotopic assay that is easier and faster to carry out than the RIAs previously described and that could be used in the clinical laboratory. We report here an ELISA for the measurement of ProT {alpha} based on the detection of thymosin {alpha}1, the N terminus of ProT {alpha}, in extracts of different human tumors and of mouse tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of tumors and tissues.
Tumors were obtained from patients who underwent definitive surgery at the Hospital General de Galicia (Santiago de Compostela, Spain) and stored at -20 °C. Skin tumors were four epidermoid (squamous) carcinomas and one melanoma. All breast tumors were invasive ductal carcinomas. Four esophagus carcinomas were epidermoid and one an adenocarcinoma. All colon carcinomas were adenocarcinomas. Patients were at different clinical stages. Mice (Charles River, Barcelona, Spain) were sacrificed and tissues were removed and stored at -80 °C.

The procedures followed were in accordance with the standards of the ethical committee of our institution.

Preparation of extracts.
Tumors and tissues were homogenized with a Polytron homogenizer (Kinematica, Littau, Switzerland) at room temperature in PBS (9 g/L NaCl, 50 mmol/L phosphate buffer, pH 7.5)-EDTA (2 mmol/L), centrifuged at 10 000g for 10 min, and the supernatant centrifuged at 15 000g for another 5 min. The final supernatant was stored at -20 °C until analysis.

Glutathione S -transferase (GST)-ProT {alpha} production and purification.
A PCR fragment of the human ProT {alpha} cDNA (2) containing the coding region with BamH1/EcoR1 flanking sites was subcloned into the pGEX-2T vector (Pharmacia Biotech, Barcelona, Spain). The upper primer was 5'-GTTGGATCCCATATGTCAGACGCA -3' and the lower primer was 5'-GAGCTCGAATTCCTAGTAGTCATCCTCGTC-3'. The PCR product subcloned into the pGEX-2T vector was sequenced with a fluorescein-labeled primer that anneals with the position 869–891 of the vector with the AutoRead Sequencing kit (Pharmacia Biotech). The reactions were run in an Automated Laser Fluorescent (ALF) DNA sequencer (Pharmacia Biotech). The sequence was confirmed by three independent results. Expression and affinity purification of GST-ProT {alpha} protein was carried out by standard procedures (18). Briefly, 100 mL of an overnight culture of Escherichia coli transformed with the recombinant plasmid was diluted 1:10 in fresh Luria broth–ampicillin and grown for 4 h at 37 °C. Then, isopropyl ß-D-thiogalactopyranoside was added to a final concentration of 0.1 mmol/L and the culture was incubated for 1 h. At the end of the incubation, bacteria were pelleted at 5000g for 10 min at 4 °C, resuspended in 25 mL of PBS containing 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and sonicated. Triton X-100 (2.5 mL) was added to the lysate and cleared at 10 000g for 5 min at 4 °C. The supernatant was incubated with 1 mL of glutathione–agarose (35 g/L in PBS) for 5 min at room temperature. The beads were washed three times with PBS containing 0.1 mmol/L PMSF and finally resuspended in a solution of 50 mmol/L Tris-HCl, pH 8.0, and 5 mmol/L glutathione. After a brief incubation, beads were pelleted and the supernatant was collected. This step was done thrice. The various supernatants obtained were stored at -80 °C and samples from the supernatants were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Those fractions containing GST-ProT {alpha} were pooled and dialyzed overnight against a solution of 50 mmol/L Tris-HCl pH 7.6, 5 mmol/L dithiothreitol, 50 mmol/L NaCl, and 0.1 mmol/L PMSF. The protein was aliquoted and stored at -80 °C.

Preparation of immunoplates.
GST-ProT {alpha} (2 mg/L in PBS) was coated onto the immunoplates (Immunomodules Maxisorp; Nunc, Roskilde, Denmark) to a final volume of 50 µL/well and incubated overnight at 4 °C. The plates were washed with 10 mmol/L (pH 7.5) PBS twice. Empty sites on the surface of the wells were blocked by addition of 50 µL/well of 30 g/L bovine serum albumin (BSA) in PBS and incubated for 2 h at room temperature. The plates were washed with PBS twice and stored at -80 °C.

ProT {alpha} antiserum.
The polyclonal antiserum to ProT {alpha} was made by injecting rabbits with synthetic thymosin {alpha}1 conjugated to keyhole limpet hemocyanin as previously described (19). This antiserum recognizes the fusion protein GST-ProT {alpha} but not GST alone as previously shown by means of a Western blot (19).

elisa procedure
Calibration curve and samples.
Calibration solutions of synthetic thymosin {alpha}1 in 30 g/L BSA-PBS (50 µL) ranging from 0.5 to 40 pmol were added to each well. Aliquots of the tissue extracts (10 µL) were added and brought to a final volume of 50 µL in 30 g/L BSA-PBS.

First incubation.
Thymosin {alpha}1 antiserum (50 µL) at a dilution of 1:125 in 30 g/L BSA-PBS was added to each well and incubated 1 h at room temperature. The plate was then washed with PBS three times.

Second incubation.
Goat anti-rabbit IgG conjugated with alkaline phosphatase (Immunotech, Marseille, France) (50 µL) at a dilution of 1:200 was added to each well, and after 1 h of incubation at room temperature the plate was washed with PBS three times and twice with diethanolamine (0.01 mol/L, pH 9.5) and MgCl2 (0.5 mmol/L). The substrate p-nitrophenyl phosphate in diethanolamine (50 µL, 1 g/L) was added to each well. After 1 h of incubation with the substrate, the absorbance in the wells was read with a Titertek Multiskan Spectrophotometer (Helsinki, Finland) at 405 nm. The use of a stopping solution, 50 µL of EDTA (0.1 mol/L) per well, avoids front-to-back differences.

Analysis.
The calibration curve and results were analyzed with the program Tablecurve (Jandel Corp., San Rafael, CA). Two different curves can fit our data with low P values: a logistic dose–response curve (P <0.01) (Fig. 1 A) and a biexponential curve (P <0.01) (Fig. 1B ).



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  		Figure 1. Calibration curve prepared with synthetic thymosin {alpha}1 (Thy a1) as described in Materials and Methods.

Two curves can fit the data: (A) logistic dose-response curve and (B) biexponential curve. Each curve is representative of 10 different experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Titration of thymosin {alpha}1 antiserum.
The antiserum to thymosin {alpha}1 was tested at various dilutions ranging from 1:10 000 to 1:50. The dilution 1:125 was considered adequate for the assay (Fig. 2 ). The best dilution for the second antiserum was 1:200 (data not shown).



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  		Figure 2. Titration of rabbit thymosin {alpha}1 antiserum.

Sera were applied in serial dilutions from 1:50 to 1:10 000. A goat anti-rabbit IgG conjugated with alkaline phosphatase and the substrate p-nitrophenyl phosphate were sequentially added to each well. The absorbance was read at 405 nm after 60 min of incubation. Values are given as the mean of duplicate wells. Values are representative of three different experiments.

Calibration of incubation time with substrate solution.
The absorbance was read at 405 nm from 10 min to 60 min. The absorbance was linear up to the last time tested (data not shown). The absorbance of the same sample assessed 1, 2, and 24 h after addition of the stopping solution did not change (data not shown), pointing out the convenience of using a stopping solution to avoid front-to-back differences.

Analytical recovery.
Possible interference in the determination of ProT {alpha} by other compounds in tissue extracts was investigated by adding 5 and 10 pmol of thymosin {alpha}1 to a tumor extract of known ProT {alpha} concentration. The recovery was defined as picomoles recovered divided by picomoles expected times 100 and was 103.4% (5 pmol added) and 107.4% (10 pmol added), respectively.

Specificity of the antiserum.
We previously found that thymosin {alpha}1 antiserum recognized both thymosin {alpha}1 and ProT {alpha} with a high specificity (19). Now we report that it shows no cross-reactivity with growth hormone (50 pmol), thyrotropin (50 pmol), prolactin (50 pmol), polyglutamic acid (50 nmol), or GST (50 pmol) (data not shown).

Precision profile and accuracy.
The inter- and intraassay variation were tested for all the concentrations in the calibration curve. The interassay variation was <15% for the concentrations 5–20 pmol (Fig. 3 A). The intraassay variation was <=10% for the concentrations 1–10 pmol (Fig. 3B ). The absorbance is linear with protein in the concentration range of 1–20 pmol/well. The limit of detection, defined as the concentration corresponding to the mean absorbance of the conjugate - 3 SD, was 0.697 pmol/well.



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  		Figure 3. (A) Interassay and (B) intraassay variation for all concentrations in the calibration curve [ranging from 0.5 to 40 pmol of thymosin {alpha}1 (Thy a1) per well (see Material and Methods)]; as expected, lower CVs were found in the central part of the curve.

Inter- and intraassay variation values are given as the mean of four and six different determinations, respectively.

Linear regression analysis of the calibration curve (protein in the concentration range of 1–20 pmol/well) yielded a {gamma}-intercept of 0.80 absorbance units (AU) and a slope of -0.269 AU/pmol; fit SE was 0.02867 and r1 was 0.968. Routinely, tumor samples, because of the high content of ProT {alpha}, were processed undiluted, diluted 1:5, and diluted 1:10. Values outside the linear range of the calibration curve were rejected.

ProT {alpha} concentrations in mouse tissues and human tumors.
To calibrate the ELISA we measured ProT {alpha} contents in mouse tissues: lung, liver, spleen, kidney, and brain. ProT {alpha} concentrations estimated by ELISA were similar to those reported previously estimated by RIA (20), being higher in spleen and lung than in low-proliferating tissues such as brain and heart (Fig. 4 B). We also measured the concentrations of ProT {alpha} in samples of various human tumors: skin, esophagus, colorectal, and breast (Fig. 4A ). ProT {alpha} concentrations assessed by ELISA were comparable although approximately twofold higher than those measured by RIA (data not shown).



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  		Figure 4. ProT {alpha} concentrations in samples from (A) various human tumors and (B) various mouse tissues.

Values are given as the mean of duplicate wells (•). This experiment is representative of three different experiments. In (B), average values found by others (20) are represented by (+).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tumor content of ProT {alpha} is a proliferation index of human breast tumors that might be used to identify patients at high risk for distant metastasis (17). Therefore, we decided to develop a simple assay easy to carry out in the clinical laboratory to determine ProT {alpha} concentrations in human tumor samples. We developed an ELISA for the measurement of ProT {alpha} in tissue extracts with a range of 0.5 to 40 pmol that is as sensitive as the RIAs reported (21)(17). The ELISA takes <4 h to be performed; it is therefore faster than the RIA, and radioactive material is not required. Moreover, ELISA total time can be reduced by using shorter incubation times with the substrate.

We used the recombinant fusion protein GST-ProT {alpha} to coat the wells because it can be produced in high amounts and is easy to isolate from E. coli lysates. We also tested the cross-reactivity of GST with thymosin {alpha}1 antiserum and found that it did not cross-react even at concentrations that were 21 times the concentration of GST used to coat the wells.

The use of thymosin {alpha}1 instead of ProT {alpha} allows less stringent conditions when handling tumor samples because of the high stability of thymosin {alpha}1 at room temperature (17). Tissue homogenates were prepared at room temperature to take advantage of the fact that ProT {alpha} rapidly undergoes proteolytic modifications that give rise to thymosin {alpha}1 (1). Therefore, we converted tissue ProT {alpha} to thymosin {alpha}1. Similar displacement curves for synthetic thymosin {alpha}1 and for serial dilutions of a tumor extract were obtained (data not shown), supporting that thymosin {alpha}1 was present in the extract. When we are assessing the concentrations of thymosin {alpha}1 we are also estimating ProT {alpha} concentrations from which it has been cleaved. There is a one-to-one correspondence between ProT {alpha} and thymosin {alpha}1. We used synthetic thymosin {alpha}1 to prepare the ELISA calibration curve, and the results are expressed as thymosin {alpha}1 equivalents .

To validate the method, we first analyzed ProT {alpha} concentrations in mouse tissues. ELISA estimations of ProT {alpha} were similar to those reported previously (20), being higher in spleen and lung than in low-proliferating tissues such as brain and heart. We tested the ProT {alpha} concentrations in samples of skin, esophagus, colorectal, and breast human tumors that were previously analyzed by RIA in our laboratory. We found that RIA and ELISA estimations were comparable although approximately twofold higher for ELISA in terms of absolute values. The reason for this difference is not clear, although it could be the result of the presence of an unknown protein that binds ProT {alpha} in tissue samples. Histone H1 has been reported to bind ProT {alpha} (22).

In summary, ProT {alpha} has been reported to be a tumor proliferation marker in breast cancer and probably in other cancers too. Here we report a simple, fast, and accurate ELISA to evaluate ProT {alpha} tissue concentrations that may be useful in the clinical laboratory.


   Acknowledgments
 
Supported in part by FISS grants (Fondo de Investigaciones Sanitarias de la Seguridad Social grants 93/0475 to F.D. and 92/0649 to F.R.) and XUGA grants (Xunta de Galicia 94/20818B to J.L.P., and Conselleria de Sanidad e Servicios Sociais. Direccion Xeral de Saude Publica, Programa de Screening de Cancer de Mama to F.D.).


   Footnotes
 
Departamento de Fisiología, Facultad de Medicina, Universidad de Santiago, 15705 Santiago, Spain.

1 Nonstandard abbreviations: ProT {alpha}, prothymosin {alpha}; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; and AU, absorbance units.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Haritos AA, Goodall GJ, Horecker BL. Prothymosin {alpha}: isolation and properties of the major immunoreactive form of thymosin {alpha}1. Proc Natl Acad Sci U S A 1984;80:1008-1011.
  2. Goodall GJ, Dominguez F, Horecker BL. Molecular cloning of a cDNA for human prothymosin {alpha}. Proc Natl Acad Sci U S A 1986;83:8926-8928. [Abstract/Free Full Text]
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  7. Eschenfeldt WH, Berger SL. The human prothymosin {alpha} gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc Natl Acad Sci U S A 1986;83:9403-9407. [Abstract/Free Full Text]
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  10. Conteas CN, Mutchnick MG, Palmer KC, Weller FE, Luk GD, Naylor PH, et al. Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: evidence for a nuclear site of action. Proc Natl Acad Sci U S A 1990;87:3269-3273. [Abstract/Free Full Text]
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  14. Sburlati AR, Manrow RE, Berger SL. Prothymosin {alpha} antisense oligomers inhibit myeloma cell division. Proc Natl Acad Sci U S A 1991;88:253-257. [Abstract/Free Full Text]
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