Novel Family of Gynecologic Cancer Antigens Detected by Anti-HIV Antibody

Objective: The reactivity of gynecologic cancer proteins with monoclonal antibody (MAb) directed against the human immunodeficiency virus I (HIV-I) was tested. Methods: Cytoplasmic and nuclear proteins, extracted from a broad range of gynecologic cancers obtained during standard surgical procedures, were tested in Western blotting with MAb 5023 developed against the amino acid sequences 308–322 of the envelope protein gp120 of HIV-I. Results: Three cell membrane proteins, Mrl20,000 (p120), Mr41,000 (p41), and Mr24,000 (p24), and one chromatin protein, Mr24,000 (p24), were detected by MAb 5023 in invasive, poorly differentiated cervical squamous-cell carcinoma; ovarian serous cystadenocarcinoma; poorly and well-differentiated endometrial carcinoma; vulvar squamous-cell carcinoma; and malignant mixed müllerian tumor. The same antigens were identified in cervical carcinoma cell line SiHa. Neither p120 nor p24 was recognized by other MAbs directed against the variable loop of gp120. Antigens p120 and p41 were undetectable in normal ovarian tissue and in biopsy samples of normal vaginal and rectal mucosa. Rectosigmoid cancer as well as colon carcinoma, lung carcinoma, and melanoma cell lines all tested negative. Conclusions: The identified antigens may represent either the products of human genes (proto-onc-ogenes) or, more likely, the products of an unknown virus specifically expressed in female cancer.

year in the United States, with the majority of these being of 3 neoplasms: carcinoma of the cervix, endometrial carcinoma, and epithelial carcinoma of the ovary. Ovarian carcinoma and breast carcinoma together account for one-third of all cancers occurring in women and are responsible for approximately one-quarter of cancer-related deaths in females. [1][2][3] Inherited forms of ovarian and breast cancer may frequently 4-6 be associated with the genetic locus BRCA17 or with mutations in other genes like p538 or Her2/erbB29 or estrogen receptors. 10 Since most gynecologic cancer patients do not exhibit any known genetic abnormalities, the etiology of these cancers remains unclear.
Due to the retroviral origin of several cancers in other vertebrates, 11 a search for cancer virus in humans was performed in several laboratories. [12][13][14] However, except for cervical cancer which is definitely linked with human papillomavirus infection, is, 16 the viral etiology of female malignancies has never been confirmed.
On the other hand, 5 0% of human immunodeficiency virus (HIV)-infected women develop cervical cancer, which has a more virulent course in HIV-infected than in noninfected women. [17][18][19][20][21][22] Immunodeficiency which follows HIV infection, was presented as an explanation for the unusually fast progression of cervical cancer in these patients, i) We report that HIV-cross-reactive antigens are expressed in several gynecologic cancers and may represent novel markers of female malignancies. Since many other cancer-associated antigens are also expressed in much smaller quantities on normal cell counterparts, the identification of novel, cancerspecific targets 23-2s is necessary for effective cancer diagnosis and immunotherapy. PBS, and tested by the light microscope. Ovarian cancer was free of erythrocytes, while cervical, endometrial, and vulvar cancer contained 4-7% of erythrocytes. The cells were homogenized in 0.3 5 M sucrose/10 mM KCI/1.5 mM MgCli/10 mM Tris-HCl (pH 7.6)/0.12% Triton X-100/12 mM 2-mercaptoethanol (approximately ml/0.5 cm 3 of the cancer tissue, with the exception of cervical cancer, which was ml/0.25 cm) and centrifuged at 600 g for 10 min. The supernatant was defined as the cytoplasmic fraction (crude, membrane-containing). In some experiments, the cytoplasm was centrifuged for another 30 min at 10,000 g to remove mitochondria and then for h at 100,000 g to obtain the microsomal (plasma-membrane) fraction. The nuclear pellet was first washed with 0.2 M sucrose/3 mM CaCl2/50 mM Tris-HC1 (pH 7.6) and then with 0.14 M NaC1/10 mM Tris-HCl (pH 8.3) and centrifuged at 700 g for 10 min. The pellet was swollen in a small amount of mM Tris-HCl (pH 7.9) and centrifuged through 1.7 M sucrose, 10 mM Tris-HC1 (pH 7), at 160,000 g for 80 min. Chromatin was pelleted at the bottom of the tube.

Gynecologic
Electrophoresis of Proteins Nuclear, chromatin, cytoplasmic, and membrane proteins were analyzed by electrophoresis in 7.5-10% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) in buffer containing 250 mM Tris-HC1 (pH 8.3), 195 mM glycine, and 0.1% SDS, according to Laemmli et al. 27 Gels were run at 100 V for [3][4] h. The molecular weights of proteins were estimated using high-range and lowrange prestained protein markers from Bio-Rad.

Monoclonal Antibodies
Monoclonal antibodies (MAbs) 5023 and 5025 (against HIV-1 gp120 amino acid region 308-322) 2s'26 are from the DuPont Company. Iodination of MAbs was routinely performed by the IO-DOGEN method, as described. 2s The specific activity of MAb was 10-20 cpm/pg.

Tissue Fractionation
The cancer and normal tissue samples obtained during surgery were extensively washed with phosphate-buffered saline (PBS) and pressed through a cell-separating mesh sieve (Sigma). Cells were centrifuged (600 g for 5 min), washed 3 times with Western Blotting Blotting of proteins from the polyacrylamide gel to the polyvinylidene difluoride (PVDF) membrane was performed in the Tris-glycine (25 mM Tris-HC1 (pH 8.6), 192 mM glycine buffer, containing 10% methanol. Transfer onto the PVDF membrane was performed at 50 V overnight. Membranes were washed with water, followed by Trisglycine buffer. Filters were incubated with 1% bovine serum albumin (BSA) for 16 h at 0C, then incubated with MAb 5023 or 5025 (2 Ixg/ml), washed with Tris-glycine buffer, and incubated with alkaline phosphatase-conjugated goat antimouse IgG for h. After being washed with the Tris-glycine buffer, the membranes were incubated with 0.1% 1-naphthyl-phosphate and Fast Red, in 100 mM Tris-HC1 (pH 9.5), 100 mM NaC1, 5 mM MgC12.

Reactivity of Gynecologic Cancer-Associated
Antigens With MAb Against HIV-I gpl20 MAb 5023 directed against HIV-I gpl20 was tested in Western blotting for reactivity with cytoplasmic, plasma-membrane, nuclear, and chromatin proteins of ovarian, cervical, endometrial, and vulvar cancer and mixed mtillerian tumor obtained during surgery. To eliminate a possibility of MAb 5023 cross-reactivity with cells other than malignant, which are present in the cancer isolated from the patient, the cervical cancer cell line SiHa was used in the same set of experiments. Three antigens were detected by MAb 5023 in the cytoplasmic/ plasma-membrane fraction of all gynecologic cancer types: high molecular weight proteins of mr120,000 (p120), Mr41,000 (p41), and Mr24,000 (p24) (Fig. 1A-C). The number of cancer samples that tested positive for particular antigens are shown in Table 1. All samples tested positive for p24 and most were also positive for p 120, p41, or both. Another MAb directed against HIV-I gpl20, MAb 5025, reacted very weakly with Mr41,000 antigen (Fig. 1D), but not with other antigens.
Cervical Cancer In a model experiment with cervical cancer cell line SiHa, the 3 antigens p120, p41, and p24 were detected by MAb 5023 in the cytoplasm and the antigens p41 and p24 in the chromatin (Fig. 1A, lanes 5, 6).
MAb 5023 reacted with all cervical cancer samples obtained from 9 different patients; however, the expression of particular antigens varied. All cancer samples were histologically analyzed and described as invasive, poorly differentiated squamouscell carcinomas. Cervical cancer CI (Fig. 1, lane 1) and CII (not shown) expressed a well-seen cytoplasmic antigen p41 and a much weaker p24, but did not express p 120. Cervical cancer sample CIII expressed only MAb 5023-reactive protein, p24 (Fig. 1, lane 2). In cancer CIV, MAb 5023 detected 2 antigens, p 120 and p24, but did not detect p41 (Fig. A, lane 3). In cancer CV (Fig. A, lane   4) and CVI, CVII, CVIII, and CIX (not shown), all 3 antigens (p120, p41, and p24) were detected. Due to the small size of the cervical cancer, separation of the nucleus from the cytoplasm was not always possible, and total extracts of cervical cancer ClV and CV (lanes 3, 4) were analyzed. However, since p120 is expressed in neither the chromatin of the SiHa cell line (Fig. 1A, lane 6) nor the chromatin of other gynecologic cancers (Fig. 1B,C), strictly cytoplasmic localization of p120 may be established. In addition to the 3 major antigens migrating in the gel with the same mobility as the antigens expressed by the cell line SiHa (Fig. 1A, lane 5), a few protein bands of lower molecular weights (<24,000) were detected in some cervical cancer samples (Fig. 1A, lanes 1, 4). Whether these low molecular weight proteins represent degradation products was not established.
Ovarian Cancer Antigen p120 was detected by MAb 5023 in the cytoplasm of all tested ovarian cancer samples (  Fig. 3, lane 1), except for cancer obtained from patient OI (Fig. 1B, lane 1) and OVI (not shown). Cancer OI was histologically characterized as poorly differentiated papillary serous adenocarcinoma involving the left fallopian tube and ovary. Ovarian cancer OVI was histologically described as mucinous adenocarcinoma. The other cancers (OIl-V) were described as typical serous cystadenocarcinomas of the ovary with the exception of OVII, which was the endometrioid carcinoma of the ovary.
Antigen p120 was undetectable in the nuclear fractions (Fig. 1B, lanes 2, 5, 7). The Mrl20,000 band visible in Figure 1B, lane 2, represents impurities attached from the cytoplasm to the nucleus during cell fractionation. After purification of the chromatin, the Mrl20,000 band was not visible (not shown). Antigen p41 was detected as a weak band in all ovarian cancer samples, but it was detected at a relatively high level in the nucleus of ovarian cancer OIII. Antigen p24 was visible in the cytoplasm and in the nucleus of all ovarian cancer samples. Similarly, as in cervical cancer (Fig. 1A), antigen p24 exhibited a clear association with both cytoplasmic and nuclear fraction (Fig. B, lanes 1-7). In the cytoplasm, an antigen of M "-'20,000 frequently   followed p24 (Fig. 1B, lanes 3, 4, 6). In the nuclear fraction, the protein of Mr20,000 was undetectable, but instead a few M < 20,000 bands were visible. Due to the low molecular weights of these bands, we cannot exclude the possibility that these bands represent the products of protein degradation.

Endometrial Cancer
In endometrial cancer EII, which according to the histologic analysis represented poorly differentiated adenocarcinoma, all 3 cytoplasmic antigens (p 120, p41, p24) and p24 in the chromatin (Fig. 1C, lanes EII-C, N) strongly reacted with MAb 5023.
The reactivity of MAb 5023 with p120 was stron- HIV-I gpl20, with cytoplasmic (C), nuclear (N), total (C+N), or chromatin (Ch) fractions isolated from particular cancer samples. The sample marked as pelvic cancer (C) was analyzed prior to the histologic examination and represents mixed millerian tumor. Proteins were separated in 7.5-12% polyacrylamide gel. Different migration is due to the different time of electrophoresis and a different gel density. Prestained protein markers (high and low molecular weight) were used in each experiment to determine the molecular weights of the antigens. Rectosigmoid cancer in C is shown as a negative control, i.e., a nongynecologic cancer. ger than with other antigens in the same tissue and also stronger than in any other tested cancer ( Fig.   1A-C). In contrast to p120, p24 was much weaker than in cervical cancer (Fig. 1A), ovarian cancer (Fig. 1B), mixed mfillerian tumor, or vulvar cancer (Fig. 1C). The second endometrial cancer, EI (Fig. C, EI), which histologically represented well-differentiated adenocarcinoma, also expressed all 3 antigens. However, the reaction of p 120 with MAb 5023 was weaker, and the reaction of p24 was stronger than in EII. Antigen p20 followed p24. The nucleus contained only p24 (not shown). Two other patients with advanced endometrial cancer (not shown) showed the same pattern as patient EII.

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY
Vulvar Cancer Vulvar cancer (invasive squamous-cell carcinoma) showed 3 protein bands (p120, p41, and p24) in the cytoplasm as well as p41 and p24 (Fig. 1C, lanes VI-C and VI-N, respectively) in the nucleus.
Cytosolic Localization of Antigen p120 The experiments shown in Figure were done with the crude, membrane-containing cytoplasmic fraction. In order to determine whether the antigens recognized by the anti-HIV-I MAb 5023 exhibit plasma-membrane or cytosolic localization, the cytoplasm isolated from the mixed mtillerian tumor of patient PIII was split into pure cytosol, microsomal, and chromatin fractions (see Methods section). Electrophoretic analysis followed by Western blotting showed that p120 was localized selectively in the cytosolic fraction (Fig. 2, lane 1), while p24 was mainly detected in the microsomal fraction and in the chromatin (Fig. 2, lanes 2, 3).

Specificity of MAb 5023 Binding to Gynecologic Cancer Antigens
Cancers other than gynecologic cancer did not react with MAb 5023. Rectosigmoid cancer, fractionated into cytoplasmic and nuclear fractions, did not react with MAb 5023 at the Mrl20,000 and Mr41,000 region. Very weak bands were detected in the cytoplasm and in the chromatin at the Mr24,000 region (Fig. 1C, R-SI). MAb 5023 also did not react with a broad range of colorectal carcinoma, lung carcinoma, and melanoma cell lines (not shown). 24 A nodule isolated from the small bowel of the patient with a Sertoli-Leydig cell tumor of the ovary, which was histologically characterized as fibrous tissue, also tested negative in Western blotting with MAb 5023 (Fig. 2, lane 4).
The normal ovarian tissue biopsy from patient OVII (Fig. 3, lane 2) did not show reactivity with MAb 5023 except for reactivity with the Mr24,000 protein, although cancer isolated from the same patient (Fig. 3, lane 1) exhibited a strong reactivity of p 120 and p41 with MAb 5023.
In general, the HIV-cross-reactive antigens p 120 and p41 were found to be specifically expressed in

DISCUSSION
MAb 5023 developed against amino acid residues 308-322 of the variable V3 loop of the HIV-1 envelope protein gpl20 was found to cross-react with antigens p120, p41, and p24 in a broad range of gynecologic cancers, including squamous-cell carcinoma of the cervix, ovarian serous cystadenocarcinoma, endometrial cancer, malignant mixed mtillerian tumor, and vulvar cancer. Antigen p120 was selectively localized in the cytoplasm, while p41 and p24 were localized in the cytoplasm and the nucleus.
In addition to the reactivity with gynecologic cancer, MAb 5023 exhibits strong binding to breast carcinoma cell antigens p 160/p80, p45, and p24. 24 Since the molecular weight of p 160 in breast cancer cells corresponds to the molecular weight of the precursor for HIV envelope proteins, and the molecular weight of p 120 in gynecologic cancer corresponds to the molecular weight of the HIV envelope antigen gp 120, an accidental homology of short epitopes is highly unlikely. Since MAb directed against I-IIV-I gpl20 is effectively internalized by breast cancer 24 and cervical cancer cells, which leads to stimulation of cell proliferation (Rakowicz-Szulczynska, unpublished data), a growth-factor receptor-like character of HIV-cross-reactive cancer antigens may be anticipated. The fact that HIVcross-reactive antigens are expressed in various gynecologic cancers of different histologic structures would support the growth-factor receptor-like character of these proteins. On the other hand, the HIV-1-cross-reactive antigens were undetectable in colorectal carcinoma, lung carcinoma, and melanoma cell lines, which makes a simple growthfactor receptor-like character of these proteins highly unlikely. We suggest that an unknown retrovirus with some homology to HIV-1 might be expressed in gynecologic cancer. Very recently, we have obtained results with the polymerase chain reaction (PCR) confirming that long regions with 100% homology to HIV-I are present in gynecologic cancer DNA (Rakowicz-Szulczynska, unpublished data).
HIV-cross-reactive antigens p120 and p41 seem to be selectively expressed by cancer cells. Normal ovarian tissue, vaginal mucosa, and colon mucosa did not express p120 or p41. It is also noteworthy that rectosigmoid cancer (Fig. 1C), as well as several melanoma, colorectal carcinoma, and lung carcinoma cell lines tested previously, 24 do not express HIV-cross-reactive antigens p 120 and p41. Thus, these antigens are specifically associated with breast 24 and gynecologic cancer. In contrast to p 120 and p41, p24 was found in some other cancer cell lines, 24 as well as in normal ovarian tissue and in noninfected lymphocytes. 25 Protein p24 cannot be considered a cancer-associated antigen.
MAb 5023 was developed against a short polypeptide (RIQRGPGRAFVTIGK), corresponding to the amino acids 308-322 of I-IIV-1 gpl20, but this MAb binds to the much shorter amino acid region GRAF. 26 It is likely that this core epitope is expressed in gynecologic cancer and breast cancer antigens. Another MAb, MAb 5025, which was developed against the same synthetic peptide but recognizes the sequences RAF, did not bind to gp 120 and p24, but reacted weakly with p41 (Fig.  1D). It is noteworthy that a fraction of human antibodies isolated from HIV-infected people also recognized in Western blotting breast cancer 24 and gynecologic cancer (unpublished data) antigens.
The results suggest that HIV-infected patients produce antibodies that cross-react with cancer. The biological function and diagnostic value, as well as the genetic origin, of the HIV-cross-reactive cancer antigens remain to be further investigated.