The P2X7 receptor: a novel biomarker of uterine epithelial cancers

Abstract

Objective: To determine expression of the P2X(7) receptor in normal and in cancer uterine tissues. The rationale was that the receptor P2X(7) regulates constitutive apoptosis in uterine epithelial cells, and previous studies showed diminished P2X(7)-mediated apoptosis in cancer uterine cells compared with normal cells.

Methods: A clinical, experimental feasibility study. Normal (n = 42) and cancer uterine tissues (n = 47) were obtained from a total of 72 women ages 25 to 75. End points for P2X(7) mRNA were quantitative PCR and in situ hybridization, and end points for P2X(7) protein were Western blots and immunostaining using anti-P2X(7) antibody.

Results: (a) In normal uteri, P2X(7) mRNA and protein were expressed predominantly in the epithelial (endometrial, endocervical, and ectocervical) cells. (b) Expression of the P2X(7) mRNA and protein was absent from endometrial and endocervical adenocarcinoma tissues and from cervical squamous cell carcinoma tissues. (c) In cervical dysplasia, P2X(7) protein was absent in the dysplastic lesions. (d) Semiquantitative analysis using P2X(7) mRNA (normalized in each tissue to the constitutive glyceraldehyde-3-phosphate dehydrogenase) and P2X(7) protein levels (normalized in each tissue to the constitutive tubulin) revealed that P2X(7) mRNA and/or protein levels can distinguish uterine normal from cancer tissues at high degrees of sensitivity (92%, 100%) and specificity (100%, 90%).

Summary and conclusions: (a) Levels of the P2X(7) are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. (b) The data suggest that tissue P2X(7) mRNA and protein levels could be used as a novel biomarker to differentiate normal and cancer uterine epithelial tissues.

Figures

Figure 1

A. Western blots using the anti-P2X7 antibody in the absence (lanes 1, 2, and 5-16) or presence (lanes 3 and 4) of the P2X7 antigen. Numbers at the top of the figures , patients’ coded identifiers. Case 120 was normal endometrium. In cases 216, 164, and 460, endometrial normal (N) and cancer (C) tissues were obtained from the same woman. Where indicated, gels were reprobed with anti-tubulin antibody. Coincubation of the anti-P2X7 antibody with the P2X7 antigen blocked expression of the 65 to 85–kDa (P2X7) and the 42 to 45 – kDa (P2X7-j) immunoreactivities. B. Immunostaining of normal endometrium with anti-P2X7 antibody in the presence or absence of the P2X7 antigen. The experiment was repeated thrice with similar trends. C. P2X7 (black columns) and P2X7-j (white columns) mRNA assayed by real-time reverse transcription-PCR using normal and cancer endometrial tissues obtained from women with the designated codes.

Figure 2

Immunostaining of endometrial (a-n), endocervical (o-z), and ectocervical (w-zv) normal (a-f, o-r, and w-zh), dysplasia (zi-zr), and cancer tissues [endometrial (g-n) and endocervical (s-v) adenocarcinomas and squamous cell carcinoma (zs-zv)] with the anti-P2X7 antibody (a, c, e, g, i, k, m, o, q, s, u, w, y, za, zc, ze, zg, zi, zk, zm, zo, zq, zs, and zu) or with the anti-E-cadherin antibody (to show the epithelial components of the tissues; b, d, f, h, j, l, n, p, r, t, v, x, z, zb, zd, zf, zh, zj, zl, zn, zp, zr, zt, and zv). zi to zl, mild cervical dysplasia; zm to zp, moderate cervical dysplasia; zq and zr, severe cervical dysplasia. a to d, o to r, and zi to zr, obtained from women ages 35 to 47; e, f, w to zb, zg, zh, and zs to zt, obtained from women ages 55 to 67. In sections containing normal endometrial glands plus endometrial cancer (k-n), the anti-P2X7 antibody stained the normal glands (k, four glands in the middle; m, two glands at the left bottom corner) but not the cancerous tissues. y, arrowhead, endocervical columnar (upward) –squamometa-plastic (downward) junction. za, arrowhead, squamometaplastic (upward) – squamous (downward) junction. ze and zf, hyperkeratosis. The experiments were repeated two to four times with similar trends. Magnification, ×4 to ×20.

Figure 3

Expression of the P2X7 mRNA: in situ hybridization. a to c, cultures of normal human foreskin keratinocytes were either not hybridized (a), hybridized with the sense P2X7 probe (b), or hybridized with the antisense P2X7 probe (c). Nuclei were counterstained as described in Materials and Methods. Specific hybridization of the P2X7 probe in c is seen as intense blue perinuclear and cytoplasmic reaction with the anti-digoxigenin antibody. The experiment was repeated twice with similar trends. d to k, tetracycline repressor–expressing Madin-Darby canine kidney cells (MDCK) were transfected with either the P2X7 cDNA or the P2X7-j cDNA, grown in the absence (Dox−) or presence (Dox+) of doxycyline, and hybridized either with the sense P2X7 probe (S) or with the antisense P2X7 probe (AS). Nuclei were not stained. g, specific hybridization of the P2X7 probe was seen only in doxycyline-treated cells transfected with the P2X7 cDNA and hybridized with the antisense P2X7 probe. The experiment was repeated twice with similar trends. l to x, in situ hybridization with the antisense P2X7 probe of cross-sections of human endometrial (l-o), endocervical (p-s), and ectocervical (t, u, w, and x) tissues. l, m, p, q, and t to v, normal tissues; n, o, r, s, w, and x, cancer tissues. Magnification, ×4 (l, n, p, r, t, v, and w) or ×20 (m, o, q, s, u, and x). v, cross-section of the normal ectocervix hybridized with the sense P2X7 probe. l, m, p, q, t, and u, specific hybridization of the antisense P2X7 probe was seen in the epithelia of the normal tissues. The experiment was repeated three to five times with similar trends.

Figure 4

Case-based analysis of tissue expression of P2X7 mRNA (top) and P2X7 protein (bottom). Endometrial, endocervical, and ectocervical tissues were obtained from the women identified with the designated codes. In some cases, normal (white columns) and cancer (black columns) tissues were obtained from the same woman. P2X7 mRNA levels in each tissue were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (determined by real-time PCR), and P2X7 protein levels were normalized to tubulin protein levels (determined by densitometry of the 65-85–kDa complex of the Western blots). AU, arbitrary units.

Figure 5

Summary of the data in Fig. 4. Individual P2X7 results for each tissue of Fig. 4 were grouped to normal (N; ○) or cancer (C; ●) mRNA or protein levels. The chosen cutoff points (horizontal lines) were as follows: positive P2X7 mRNA, ≥1,000; positive P2X7 protein, ≥15. Data were statistically analyzed in Table 1A and B.