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Centre of Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Queensland 4001, Australia [Y. D., A. K., L. B., J. A. C.]; Prince Henry’s Institute of Medical Research, Clayton, 3168 Australia [S. C., P. J. F.]; Department of Obstetrics and Gynecology, Royal Women’s Hospital, Herston, Brisbane, Australia [J. N.]; and Department of Pathology, Royal Brisbane Hospital, Herston, Brisbane, 4029 Australia [H. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR, Southern Blot, and DNA Sequencing Analysis.
Total RNA was isolated from tumor cells or tissues using TRIzol reagent (Life Technologies, Inc.) following the manufacturer’s instructions. Two µg of total RNA was reverse transcribed into first-strand cDNA using Superscript II in a 20-µl reaction. Fifty ng of KLK4-specific primers (5'-GCGGCACTGGTCATGGAAAACG-3' and 5'-CAAGGCCCTGCAAGTACCCG-3') and Platinum Taq (Life Technologies, Inc.) were used for PCR. PCR was performed with 1 µl of cDNA for three different ovarian samples (NOE cells, SER cancer cells, and the OVCAR-3 cell line) for 30, 35, 40, and 45 cycles to determine that amplification was in the linear range (Fig. 1A)
. The final chosen optimum cycling conditions were 94°C for 5 min, followed by 40 cycles of 94°C, 62°C, and 72°C for 1 min each, and a final extension at 72°C for 7 min. PCR for ß2-microglobulin (5'-TGAATTGCTATGTGTCTGGGT-3' and 5'-CCTCCATGATGCTGCTTACAT-3'), which was used as an internal control, was performed for 35 cycles with similar PCR conditions except for the annealing temperature (56°C). The PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. The resulting amplicons were analyzed by Southern blot hybridization using a DIG 3' end-labeled KLK4 oligonucleotide probe (5'-CTCCTACACCATCGGGCTGGGC-3') in Easyhyb solution (Roche, Mannheim, Germany) overnight at 37°C. Washes with 0.2 x SSC/0.1% SDS were performed at 37°C. The membrane was blocked with anti-DIG antibody, and signals were detected by CDP-star (Roche) using X-ray film. The intensity of bands was determined by densitometry (GS-690 Imaging; Bio-Rad) using the Bio-Rad Multi-Analyst program. PCR products were also gel purified (Qiagen Pty, Ltd., Hilden, Germany) and sequenced. DNA sequences were analyzed using tBLASTN.
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Immunohistochemistry.
Immunohistochemical staining was performed on sections obtained from the same tissue blocks as above using a Zymed kit (Zymed Laboratories, Inc., San Francisco, CA). Paraffin sections (4 µm) were deparaffinized, and antigen retrieval was performed by microwave heat treatment in 10 mM sodium citrate buffer (pH 6.0). After H2O2 treatment and blocking, the sections were incubated (2 h) with a combination of affinity-purified anti-hK4 peptide antibodies (1/250 dilution) at room temperature, then biotinylated goat antirabbit immunoglobulins and streptavidin-horseradish peroxidase conjugate following the manufacturer’s instructions. The hK4 antibodies were generated by immunization of New Zealand rabbits with peptides from three different regions of the hK4 protein, respectively. The production, specificity, and characterization of the hK4 antibodies will be described elsewhere4
Peroxidase activity was detected using 3,3'-diaminobenzidine (Sigma Chemical Co.) as the chromogen with H2O2 as the substrate. The sections were counterstained with Mayer hematoxylin. Negative controls were performed by using normal rabbit serum or primary antibody preabsorbed with hK4 peptides instead of the primary antibody.
Western Blot Analysis.
Cytoplasmic extracts (150 µg of protein) from cultured tumor cells were electrophoresed on 12% SDS-polyacrylamide gels, and the protein was then transferred to a Protran membrane (Schleicher & Schüll, Dassel, Germany). The membrane was stained with ponceau S (Sigma Chemical Co.) to determine equivalent protein loading in each lane, blocked with 5% skim milk in Tris-buffered saline/Tween 20 overnight at 4°C, and then incubated with the same affinity-purified anti-hK4 peptide antibodies as used above (1/500 dilution, 2 h) at room temperature. The blot was washed and then incubated (1 h) with a horseradish peroxidase goat antirabbit IgG (Dako, Glostrup, Denmark; 1/2000 dilution) at room temperature. The signals were visualized on X-ray film by enhanced chemiluminescence. Densitometry analysis was performed to determine any changes in signal intensity on the Western blot.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and indicate that 40 PCR cycles are within the linear amplification range. Thus, six normal ovaries, 31 different ovarian tumors, and eight SER ovarian cancer cell lines were examined for their expression of KLK4 by RT-PCR over 40 cycles, Southern blot, and densitometry analyses. The results of Southern blot analysis of the KLK4 expression pattern in representative samples are shown in Fig. 1B
with the densitometry analysis of the Southern blot for these samples shown in Fig. 1D
. ß2-microglobulin, which was used as an internal control (Figs. 1C
and 2B
), showed a consistent pattern of expression in all samples indicating the integrity of the RNA. Clinical information of all tumor tissues and cell lines are summarized in Table 1
. KLK4 expression was detected in normal ovaries (four of six), as well as epithelial-derived SER (benign: two of two; malignant: 11 of 11; cell lines: eight of eight), MUC (benign: one of one; malignant: two of three), END (four of five), clear cell tumors (two of three), and GCTs (three of six; Table 1
). The level of KLK4 expression was determined from the absorbance (A) reading of bands obtained from densitometry analysis. A higher level of KLK4 expression (>40 A) was observed in the two benign adenomas, 10 of 11 SER carcinomas, and all eight SER-derived cancer cell lines compared with the normal ovaries, MUC, END, and clear cell and GCTs that are KLK4 positive (Fig. 1, B and D
and Table 1
). Of these latter tumor types or normal ovaries, only four exhibited a level of KLK4 expression >40 A, and indeed, KLK4 expression was not observed in many of these samples, although 40 cycles of amplification were performed. These results suggest that KLK4 is most highly expressed by late stage SER epithelial ovarian carcinomas.
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and 2A
). These three KLK4 variants were noted in all SER epithelial ovarian tumors, including SER adenomas (two of two), SER carcinomas (11 of 11), and SER carcinoma cell lines (eight of eight), whereas three of four MUC tumors, four of five END carcinomas, two of three CCCs, and only one of six GCTs showed expression of these variants (Fig. 1
and Table 1
). To examine the three alternate forms of KLK4, PCR products from NOE cells, primary cultured cells from a SER ovarian carcinoma and from the ovarian cancer cell line OAW42 (Fig. 2A)
, were sequenced. Three alternate sequences (variants 1–3 in Fig. 2A
) were successfully identified. Variant 1 (Fig. 2A
, 609 bp) has an 83-bp insertion of intronic sequence from intron 3, and variant 2 (401 bp) has a 12-bp insertion of intronic sequence from intron 2 and exon 4 deletion. The variant 2 sequence is similar to a variant wi2th 2 12-bp insertions reported in prostate tissue (19)
but also includes an exon 4 deletion. Variant 3 (389 bp) has only the region corresponding to exon 4 deleted (Fig. 2C)
and is similar to the sequence reported previously in an endometrial carcinoma cell line (14)
. All these variants exhibit a frame shift of the coding region that generates a premature stop codon giving rise to a truncated protein product that does not contain the Asp116, variant 2 only or Ser residue (Ser207) of the catalytic triad (Fig. 2C)
. This result was confirmed by RT-PCR using DNase I-treated RNA, indicating that this finding is not attributable to genomic DNA contamination.
Expression of KLK4 Transcripts and hK4 in Ovarian Cancer Tissues.
The in situ hybridization and immunohistochemistry results shown in Fig. 3, A–E
are representative of the patterns observed for the two normal ovaries and all four ovarian cancer samples. On in situ hybridization with a DIG-labeled KLK4 antisense cRNA probe, KLK4 expression was detected in the ovarian adenocarcinoma cells of moderately differentiated SER carcinomas (Fig. 3A)
. Ovarian carcinoma sections hybridized with a KLK4 sense cRNA probe were negative (Fig. 3B)
. On immunohistochemistry, using a combination of the affinity-purified hK4 antipeptide antibodies, essentially no hK4 staining was observed in the normal ovary (Fig. 3C)
. In SER ovarian carcinoma cells, hK4 staining was primarily found in the cytoplasm, with some focal membrane localization observed (Fig. 3D)
, whereas only focal and equivocal staining was seen in stroma. No staining was seen in the negative control with normal rabbit serum instead of the primary antibody (Fig. 3E)
or preabsorption of the hK4 antibodies with K4 peptides (data not shown).
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40,000 in the prostate cancer cell line LNCaP, eight different ovarian cancer cell lines (see "Materials and Methods"), and primary cultured ovarian carcinoma cells. Fig. 3F
(Lanes 1–4) shows a representative Western blot with one hK4 antipeptide antibody (COOH-terminal directed) of the ovarian cancer cell line, OAW42, and primary cultured SER ovarian carcinoma cells, N12 and N15, with LNCaP as the positive control. Similar expression patterns were observed with the other two antibodies to peptides from different regions of the hK4 protein (Fig. 3F
, Lanes 10 and 11), although these bands were fainter as these antibodies are not as avid. The estrogen receptor-positive ovarian carcinoma cell line OVCAR-3 was used to evaluate if hK4 expression is under estrogen regulation. As shown in Fig. 3E
(Lanes 5–9), a 1.5–4-fold up-regulation of hK4 intracellular levels by 100 nM estrogen was found, and this regulation was time dependent (Fig. 3G)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40,000 and report that the intracellular level of hK4 is up-regulated by estrogen treatment in the estrogen receptor-positive ovarian carcinoma cell line OVCAR-3. Previous studies have shown that KLK6, KLK7, and KLK8 are overexpressed by ovarian carcinomas compared with normal ovaries (3, 4, 5, 6 , 20) . In addition, the expression of KLK5 was reported recently to be associated with poor prognosis of ovarian cancer patients (21) . We now report a similar pattern of expression for the fourth KLK gene, KLK4, in benign and malignant ovarian tissues. In contrast, it is interesting to note that Bicher et al. (22) reported that loss of heterozygosity on chromosome 19q13.2–q13.4, a region spanning the human KLK gene locus, was present in 53% of ovarian cancers. Indeed, the expression of another KLK gene, KLK10 (normal epithelial cell-specific 1 gene NES1), is down-regulated in breast cancer (23) . These data demonstrate that this region is an important area of genomic activity with respect to hormone-dependent cancers.
In the present study, using RT-PCR, Southern blot, and densitometry analyses, we have shown that the expression of KLK4 and its variants may be related to the histology and/or stage of ovarian tumors. RT-PCR analysis, performed for 40 amplification cycles, showed clear expression in many samples compared with no expression in other samples, allowing a comparative analysis. All of the stage III and stage IV SER ovarian carcinomas showed the highest KLK4 expression (as indicated by the intensity of the hybridization signal), whereas only four of eight of the MUC or GCTs showed high KLK4 expression, although all of the MUC tumors and five of six GCTs used in this study were early stage tumors. Indeed, three of six GCT samples showed no KLK4 signal, although they were clearly positive for ß2-microglobulin. Both adenomas also exhibited a higher level of KLK4 expression, although these findings will need to be confirmed on a larger group of samples. All of the ovarian cancer cell lines were also epithelial derived from late stage SER carcinomas and showed high KLK4 expression. These cell lines covered a spectrum from well to poorly differentiated, but no correlation between KLK4 expression and differentiation state could be drawn from this study. However, MUC ovarian tumors and GCTs have relatively reduced proliferative rates, when compared with SER tumors, and therefore, the expression of KLK4 may be related to the proliferative status of a tumor. In addition, although all late stage ovarian cancers have poor outcomes, the prognosis of early stage SER and CCCs is worse than MUC, END, and GCTs. In this context, it is of interest to note that one stage III SER carcinoma (number 16, Table 1
), three END carcinomas (numbers 32, 35, and 36, Table 1
), and two CCCs (numbers 37 and 38; Table 1
) had a better survival than the other tumors, and these tumors did not show high KLK4 expression.
We also observed that three KLK4 variants were detected in different ovarian tumors but not in normal ovaries. These variants had premature stop codons that would lead to a truncated hK4 protein if translated. All of these variants would not contain Ser207 of the catalytic triad (indeed, variant 2 would also not contain Asp116 of the catalytic triad), and therefore, they are unlikely to encode proteins with enzymatic activity. Previous studies from our laboratory have identified the KLK4 variant 3 mRNA splice form in endometrial carcinoma cell lines (14) . A similar variant to our variant 2 with a 12-bp insertion has also been reported in the prostate (19) . Moreover, mRNA variants have been demonstrated for other KLKs, such as KLK1 (24) , KLK2 (25) , KLK3 (26 , 27) , and KLK13 (KLK-L4; Ref. 28 ). Thus, variant mRNA transcripts are a common feature of the human KLK family. Overall, we have shown increased expression of the wild-type KLK4 transcript in late stage ovarian tumors and that several KLK4 mRNA variants are expressed by ovarian tumors but not by normal ovaries. It will be important to now determine whether, like PSA in prostate and breast cancer (7 , 25) , KLK4/hK4, or the KLK4 variant forms, could be a useful diagnostic or prognostic marker for some ovarian cancers or monitor this disease.
In situ hybridization and immunohistochemical staining of ovarian tumor sections from four different patients revealed that KLK4 mRNA and the hK4 protein are detected in the cytoplasm and occasionally on the cell membrane of the SER epithelial-derived adenocarcinoma cells of the tumor tissues. However, no hK4 immunostaining was observed in normal ovaries. Consistent with our immunohistochemical staining results, the cell lysates from the ovarian carcinoma cell lines and carcinoma cells showed immunoreactivity to the hK4 antibody. The difference between the Western blot determined molecular weight (Mr 40,000) and predicted (Mr 30,000) molecular weight is probably because of a post-translation modification, as the predicted hK4 amino acid sequence contains N-glycosylation sites. Given the consistent results with three antipeptide antibodies to three different regions of the hK4 protein (Fig. 3E)
, it is likely that these antibodies are detecting the endogenous hK4 protein. The cell membrane staining was a surprising finding, as other KLKs, such as PSA, are secreted enzymes and usually localized to the cytoplasm. However, there are five predicted myristoylation sites in the hK4 sequence that may indicate a cell membrane function.
KLK4 expression was found previously in the breast cancer cell line BT-474 (11) and endometrial carcinoma cell lines (14) , as well as in the prostate cancer cell line LNCaP (10) . Moreover, KLK4 mRNA expression was up-regulated by androgen, progestin, and estrogen (10 , 11) , and the intracellular levels of hK4 were induced by estrogen and progestin treatment (14) in these cell lines. Consistent with these findings, the present study showed that hK4 protein levels were induced by estrogen in the estrogen receptor-positive ovarian carcinoma cell line OVCAR-3 and that the induction was time dependent. Ovarian tumors (>50%) were found to be estrogen and progesterone receptor-positive, with some authors suggesting that SER and END tumors are more frequently positive (29) . Although it is still controversial as to the possible role of estrogen or progesterone regulation in ovarian tumorigenesis, there is some suggestion that a subgroup of patients with aggressive ovarian tumors refractory to conventional chemotherapy may benefit from hormonal therapy (29) . It will be interesting to determine whether any association exists between estrogen receptor status and KLK4/hK4 expression in advanced SER and/or other ovarian epithelial tumors.
The function of hK4 is not yet known, but hK4 shows 72% protein identity to pig enamel matrix seine proteinase 1, which degrades the ECM in preparation for enamel maturation (30) . This suggests that the function of hK4 may be similar to enamel matrix seine proteinase 1, and hK4 may be involved in the degradation of ECM. Therefore, hK4 may play a similar role to other KLKs and be involved in the progression and metastasis of several cancers. Both KLK 2 protein (hK2; Ref. 31 ) and PSA can degrade the ECM glycoprotein fibronectin (32) , and PSA can also degrade laminin (32) . PSA degrades insulin growth factor binding protein-3 and activates the pro-forms of epidermal growth factor, nerve growth factor, and transforming growth factor-ß, thus regulating the bioavailability of these growth factors (25) . Whether KLK4 will perform similar function(s) is yet to be elucidated, but a role in ECM degradation and/or growth factor activation would be consistent with its expression in the highly proliferative and invasive late stage SER carcinomas.
In summary, the expression patterns observed in the present study suggest that KLK4 mRNA and its variant forms are highly expressed in late stage ovarian cancer, particularly SER carcinomas. Additional studies are required to determine the precise function and role of KLK4 in ovarian tumorigenesis and its usefulness in the diagnosis and/or monitoring of these tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Queensland University of Technology Re-entry Fellowship for Women and the National Health and Medical Research Council of Australia. 
2 To whom requests for reprints should be addressed, at the Centre of Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, P. O. Box 2434, Brisbane, Queensland 4001, Australia. Phone: 617-3864-1899; Fax: 617-3864-1534; E-mail: j.clements{at}qut.edu.au
3 The abbreviations used are: KLK, kallikrein; hK4, KLK4 protein product; NOE, normal ovarian epithelial; SER, serous; MUC, mucinous; END, endometrioid; CCC, clear cell carcinoma; GCT, granulosa cell tumor; DIG, digoxigenin; RT-PCR, reverse transcription-PCR; ECM, extracellular matrix; Asp, aspartic acid; Ser, serine; PSA, prostate-specific antigen.
4 T. Harvey, Y. Dong, J. Hooper, and J. A. Clements. Production and characterisation of antipeptide kallikrein 4 antibodies, manuscript in preparation.
Received 12/28/00; revised 5/15/01; accepted 5/23/01.
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150 µg of protein) from the ovarian cancer cell line (OAW42), primary cultured SER ovarian carcinoma cells (N12 and N15), prostate cancer cell line LNCaP, and ß-estradiol treatment on the ovarian cancer cell line OVCAR-3. Lanes 1–9, anti-COOH-terminal peptide antibody; Lane 10, anti-NH2-terminal peptide antibody; and Lane 11, anti-mid region peptide antibody. The size of the protein molecular weight markers is shown at the left. A hK4 protein of Mr