[CANCERRESEARCH57,2651—2656.July1, 19971 Expression of Human Prostate-specific Glandular Kallikrein Protein (hK2) in the Breast Cancer Cell Line T47.D1 Ming-Li Hsieh, M. Cristine Charlesworth, Marcia Goodmanson, Shaobo Zhang, Thomas Seay, George G. Klee, Donald J. Tindall, and Charles Y. F. Young@ Departments of Urology (M-L H., S. Z, T. S., D. J. T., C. Y. F. Y.], Biochemistry and Molecular Biology [M. C. C., D. J. T., C. Y. F. Y.j, and Laboratory Medicine IM. G.. G. G. K.], Mayo Graduate Schools, Mayo Clinic/Foundation, Rochester, Minnesota 55905 ABSTRACT whether PSA plays a physiological role in these tissues in women remains to be elucidated. Human glandular kallikrein (hK2) protein, like prostate-specific anti The hK2 is another member of the human kallikrein gene family gen (PSA), is produced mainly In prostatic epithellum. It may be useful as (16). The hK2 gene was first discovered in 1987 (17). However, its a new diagnostic indicator for prostate cancer. Recently, a number of hK2-speciflc monoclonal antibodies have been developed that enable us to complete cDNA was not isolated until 1992 (18). Studies of hK2 detect hK2 protein In human prostate tissue, seminal fluid, and scm. mRNA indicate that hK2 is specific for prostatic tissue and is regu Whether hK2 can be expressed, like PSA, in nonprostatic cells is not lated by androgens via the androgen receptor (18—20).To define the known. In this study, we have characterized the presence of hK2 in an biological role and potential clinical utility of hK2 protein for prostate androgen-responsive breast cancer cell line T47-D at both the protein and cancer, recombinant hK2 proteins and hK2 peptides have been syn mRNA levels with an immunoassay, Western blot analysis, Northern blot thesized and used to generate monospecific antibodies (mAbs; Ref. analysis, and the reverse tmanscription-PCR. 21). A mAb HK1AS23, specific for hK2, has been used to detect hK2 Using a sensitive lmmunoassay with monoclonal antibodies to hK2, we protein in spent media from the androgen-dependent prostate cancer found that T47-D cells could be induced with androgens, minemalocorti cell line, LNCaP, and in human prostate tissues by Western blot coids, glucoeerticoids, and progestins to produce significantly more hK2 analysis and immunohistochemistry (22, 23). More recently, other than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction high affinity mAbs have been developed for detecting hK2 protein in of hK2 in T47-D cells was dose dependent. More interestingly, we found serum (24—26). that the hK2 in androgen-induced T47-D cell spent media appears to be The similarities of PSA and hK2 suggest that hK2 might also be the pro-form of hK2 rather than mature hK2. produced by cells other than those of prostatic origin. We demonstrate Our study demonstrates that hK2, a serine protease thought to be in the current work that hK2 can be produced at a higher level than found only in prostate-related tissues and fluids, is also produced in a PSA by a breast cancer cell line T47-D under androgen stimulation. breast cancer cell line T47-D after steroid stimulation. This finding sug This is the first study to our knowledge that demonstrates the produc gests that hK2 may have a potential role in breast cancer as well as tion of hK2 protein by nonprostate cells. This finding further under prostatic cancer and will be the Impetus for further studies of hK2 scores the similarities between breast and prostate epithelial tissue and distribution and function. may extend the potential applications of hK2 as a biomarker for breast tumor in addition to that of prostate cancer. INTRODUCTION PSA3 is a kallikrein-like renne protease that was thought to be MATERIALS AND METHODS exclusively produced by epithelial cells lining the acini and ducts of the prostate gland (1—3).Because of its tissue specificity, PSA has Materials. mAbs were prepared as described by Grauer et a!. (22) and been widely used as a marker for diagnosis and monitoring of prostate Kumar et a!. (25). HK1AS23, HK1H449, and HK1H599 are specific for hK2, and HK1H464 is specific for the pro-region of phK2. The pro-region of phK2 cancer because the introduction of PSA testing in 1986 (4, 5). How contains a seven-amino acid extension at the NH2 terminus of mature hK2 (or ever, the tissue specificity has been questioned in several recent hK2). Recombinant hK2 and phK2 were expressed and purified using the reports. A number of studies have demonstrated the presence of PSA adenovirus-induced AV12 hamster tumor cell line (25). PSA was purified from in the periurethral and perianal glands (6—8).It has been demon seminal fluid according to the procedure of Sensabaugh and Blake (27) and stated that the steroid hormone receptor-positive breast carcinoma provided by Dr. Roger Sokoloff (Hybritech, Inc., San Diego, CA). cell line T47-D can be stimulated by androgens, progestins, miner Cell Cultures and Steroid Treatments. Several human cell lines (ob alocorticoids, and glucocorticoids to produce PSA (9). More recently, tamed from American Type Culture Collection, Rockville, MD) including using a newly developed ultrasensitive PSA assay, it has been re T47D (breast), ZR75—l(breast), Hs5787 (breast), BT-20 (breast), MCF-7 ported that 30% of female breast tumor cytosolic extracts contained (breast), OVCAR-3 (ovary), Hep-G2 (liver), HT-27 (colon), and LNCaP PSA immunoreactivity, accounting for greater than 0.03 ng/mg of (prostate) were propagated in Coming 24-well culture dishes or Tl75 culture total protein (10, 11). Also, a study has shown that PSA may possibly flasks with RPM! 1640 supplemented with 5% FCS at 37°Cand 5% CO2. After reaching approximately 80% confluency, cells were incubated in phenol be used as a prognostic marker for breast cancer (12). Moreover, low red-free, serum-free RPMI 1640 for 24 h to deplete undesired steroids prior to levels of PSA were found in normal breast, normal endometrial tissue, experiments. milk of lactating women, and amniotic fluid (13—15). However, The initial stimulation experiment was performed by adding 11 different steroids to androgen receptor-positive T47-D cells. Ethanol was used as a Received 1/1(997;accepted 4/29/97. solvent to dissolve the steroids. Equivalent amounts of solvent were added to Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage control wells. Steroids were replenished every 24 h. After a 4-day incubation, charges. This article must therefore be hereby marked advertisement in accordance with spent medium was harvested for PSA and hK2 quantitation by an immuno 18 U.S.C. Section 1734 solely to indicate this fact. I Supported in part by the NIH Grants CA70892 and DK41995. metric assay as described below. Cell density was assayed by incubating cells 2 To whom requests for reprints should be addressed, at Mayo Clinic, 200 First Street, with 150 @.dof3-[4,5-dimethylthiazol-2-ylj-2,5-diphenyl-tetrazolium bromide SW, Guggenheim Building, l7-1742B, Rochester, MN 55905. (Sigma Chemical Co., St. Louis, MO; 5 mg/mI in PBS) in I ml of RPMI 1640 3 The abbreviations used are: PSA, prostate-specific antigen; mAb, monoclonal anti per well at 37°Cfor4 h. The insoluble product of 3-[4,5-dimethylthiazol-2- body; hK2, human glandular kallikrein; phK2, prohK2; DHT, dihydrotestosterone; RT PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; yll-2,5-diphenyl-tetrazolium bromide retained in the cells was then dissolved ER, estrogen receptor; AR. androgen receptor. in DMSO (Sigma) for absorbance measurement at 570 nm. The concentrations 2651 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. EXPRESSIONOFhK2 IN BREASTCANCERCELLS of PSA and hK2 were normalized by the above cell density measurements and conditions for each cycle were as follows: denaturation at 94°Cfor 60 s, expressed as nanograms/milliliter/A570. For dose effect, the same procedure annealing at 58°C (for PSA) or 60°C (for hK2) for 90 s, and DNA polymer was also performed on T47D cells after treatment with various DHT concen ization at 72°Cfor90 s. After PCR cycling, the reactions underwent DNA trations from 0.2 to 1000 nM. Similarly, the time course effects of 200 nM DHT extension at 72°Cfor 10 mm. An aliquot of PCR reaction product was used for stimulation on expression of PSA and hK2 were also examined. 1% agarose gel electrophoresis. PCR DNA products in agarose gel were To test whether different human cell lines as listed above can produce PSA visualized by ethidium bromide staining. DNA bands corresponding to PSA and hK2 proteins, these cells were incubated in the presence or absence of a and hK2 were eluted from the gel, directly subcloned into pCR-II plasmid nonmetabolizable steroid, mibolerone, for 7 days. Finally, experiments were (Invitrogen, San Diego, CA), and transformed into Escherichia coli to obtain conducted to test whether hK2 induction by androgens in T47D cells can be enough DNA for sequencing. blocked by estrogens. The experiments were performed by stimulating the cells Northern Blot Analysis. Steroid-depleted T47-D cells were cultured in with 50 nM DHT in the presence of different concentrations of estradiol. The serum-free RPM! 1640 with the addition of DHT (100 nM) for 28 h. Total RNA spent media were harvested 4 days later. was extracted by the acidic phenol-chloroform-guanidinium thiocyanate Immunoassays for PSA and hK2 Quantitation. PSA levels in culture method (18). Equal amounts of RNA (30 j.&gflane)werefractionated in the supernatants were determined by an immunoenzymatic assay using the Tan presence of ethidium bromide by denaturing gel electrophoresis and trans dem-E PSA kit (Hybritech, Inc., San Diego, CA).