Human kallikrein 14: a new potential biomarker for ovarian and breast cancer

Human kallikrein gene 14 (KLK14) is a recently discovered member of the tissue kallikrein family of secreted serine proteases, which includes hK3/prostate-specific antigen, the best cancer biomarker to date. Given that KLK14 is hormonally regulated, differentially expressed in endocrine-related cancers, and a prognostic marker for breast and ovarian cancer at the mRNA level, we hypothesize that its encoded protein, hK14, like hK3/prostate-specific antigen, may constitute a new biomarker for endocrine-related malignancies. The objective of this study was to generate immunological reagents for hK14, to develop an ELISA and immunohistochemical techniques to study its expression in normal and cancerous tissues and biological fluids. Recombinant hK14 was produced in Pichia pastoris, purified by affinity chromatography, and injected into mice and rabbits for polyclonal antibody generation. Using the mouse and rabbit antisera, a sandwich-type immunofluorometric ELISA and immunohistochemical methodologies were developed for hK14. The ELISA was sensitive (detection limit of 0.1 micro g/liter), specific for hK14, linear from 0 to 20 micro g/liter with between-run and within-run coefficients of variation of <10%. hK14 was quantified in human tissue extracts and biological fluids. Highest levels were observed in the breast, skin, prostate, seminal plasma, and amniotic fluid, with almost undetectable levels in normal serum. hK14 concentration was higher in 40% of ovarian cancer tissues compared with normal ovarian tissues. Serum hK14 levels were elevated in a proportion of patients with ovarian (65%) and breast (40%) cancers. Immunohistochemical analyses indicated strong cytoplasmic staining of hK14 by the epithelial cells of normal and malignant skin, ovary, breast, and testis. In conclusion, we report the first ELISA and immunohistochemical assays for hK14 and describe its distribution in tissues and biological fluids. Our preliminary data indicate that hK14 is a potential biomarker for breast and ovarian cancers.     

Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein (hK) gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). KLK5 was shown to be differentially expressed at the mRNA level in breast and ovarian cancer. Until now, detection of hK5 protein in either biological fluids or tissues has not been described due to lack of suitable reagents and methods. The aim of this study was to develop immunological reagents and a sensitive and specific fluorometric immunoassay (ELISA) for hK5, to examine the presence of hK5 in human tissues and biological fluids, and to study the possible clinical utility of hK5 as a biomarker for endocrine-related malignancies. Recombinant hK5 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK5 antibodies. A sandwich-type microplate immunoassay (ELISA) was developed using these antibodies, coupled with a time-resolved fluorometric detection technique. The ELISA assay was then used to measure hK5 in various biological fluids, tissue extracts, and serum samples from normal individuals and patients with various malignancies. The hK5 ELISA immunoassay has a lower detection limit of 0.1 micro g/liter, is specific for hK5, and has no cross-reactivity with other homologous kallikreins. The dynamic range is 0.1-25 micro g/liter, and within-run and between-run coefficients of variation within this range are <10%. hK5 is found in many tissues, with the highest expression levels seen in the skin, breast, salivary gland, and esophagus. hK5 is present at relatively high levels in milk of lactating women. Whereas the levels of hK5 are almost undetectable in serum of normal individuals (male and female) and patients with diverse malignancies, higher concentrations were found in a proportion of patients with ovarian (69%) and breast (49%) cancer. High levels were also detected in ascites fluid from metastatic ovarian cancer patients and in ovarian cancer tissue extracts. In conclusion, we report development of the first immunofluorometric assay for hK5 and describe the distribution of hK5 in biological fluids and tissue extracts. Our preliminary data indicate that hK5 is a potential biomarker in patients with ovarian and breast cancer.     

Human kallikrein 8, a novel biomarker for ovarian carcinoma

Human kallikrein 8 (hK8; neuropsin) is a serine protease and new member of the hK family. The aim of this study was to examine if hK8 may serve as a novel cancer biomarker. An hk8-ELISA, developed in-house, was used to study the distribution of hK8 in various biological fluids and tissue extracts from healthy individuals and ovarian cancer patients of different stages of the disease (International Federation of Obstetrics and Gynecology II-IV). For ovarian cancer patients, very high levels in ascites fluid were observed (相似文献   

The usefulness of serum human kallikrein 11 for discriminating between prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA) is the most useful tumor marker for diagnosis and monitoring of prostate cancer (CaP). Recently, we developed a specific immunoassay for human kallikrein 11 (hK11), one of the kallikrein gene family members, and found that hK11 was highly expressed in prostatic tissue and could be detected in seminal plasma (E. P. Diamandis et al., Cancer Res., 62: 295-300, 2002). The aim of this study was to investigate whether serum hK11 levels could be used to discriminate CaP from benign prostatic hyperplasia (BPH). We analyzed for hK11, total PSA, and percentage of free PSA, 150 serum samples from men with histologically confirmed BPH (n = 64) or CaP (n = 86). Total and free PSA levels were measured by the Immulite PSA assay, and hK11 levels were measured by our previously published immunofluorometric assay. Serum hK11 levels and the hK11:total PSA ratio were both significantly lower in CaP patients than in BPH patients. In the subgroup of patients with percentage of free PSA less than 20, an additional 54% of BPH patients could have avoided biopsies by using the hK11:total PSA ratio. Receiver operating characteristic (ROC) curve analysis demonstrated that the hK11:total PSA ratio [area under the curve (AUC), 0.83] and percentage of free PSA (AUC, 0.83) were much stronger predictors of CaP than total PSA (AUC, 0.69). These preliminary data suggest that the hK11:total PSA ratio could be a useful tumor marker for CaP and could be combined with percentage of PSA to further reduce the number of unnecessary prostatic biopsies.     

Expanded human tissue kallikrein family--a novel panel of cancer biomarkers.

The full characterization of the human kallikrein gene locus has allowed identification of all members of this gene family on chromosome 19q13.4 and the establishment of common structural criteria, at both the mRNA and protein level. The human kallikrein gene family now consists of 15 members; their mRNA and protein structure, tissue expression and hormonal regulation patterns have been delineated. In addition to prostate-specific antigen (PSA, hK3), which is an established tumor marker for prostate cancer diagnosis and follow-up, and human glandular kallikrein (hK2), an emerging prostate cancer biomarker, accumulating evidence indicates that many other members of the human kallikrein gene family are also implicated in endocrine-related malignancies. Many kallikreins are differentially regulated in breast, prostate, ovarian and testicular cancers. In addition, preliminary reports indicate that three newly identified kallikreins (hK6, hK10 and hK11) are serum biomarkers for diagnosis and monitoring of ovarian and prostate cancer. The mechanism by which kallikreins might be involved in the pathogenesis and/or progression of cancer is not as yet fully understood. Preliminary reports indicate a possible role of kallikreins in controlling vital processes, like apoptosis, angiogenesis and tumor metastasis by cleavage of critical substrates such as growth factors, hormones or extracellular matrix. In this review, we present data on the differential expression of kallikreins in cancer at both the mRNA and protein levels, and propose future directions of research towards our understanding of the involvement of kallikreins in cancer and their possible diagnostic, prognostic and therapeutic applications.     

Human tissue kallikrein gene family: applications in cancer

Human tissue kallikrein genes, located on the long arm of chromosome 19, are a subgroup of the serine protease family of proteolytic enzymes. Initially thought to consist of three members, the human kallikrein locus has now been extended and includes 15 tandemly located genes. These genes, and their protein products, share a high degree of homology and are expressed in a wide array of tissues, mainly those that are under steroid hormone control. PSA (hK3) is one of the human kallikreins, and is the most useful tumor marker for prostate cancer screening, diagnosis, prognosis and monitoring. hK2, another prostate-specific kallikrein, has also been proposed as a complementary prostate cancer biomarker. In the past 5 years, the newly discovered kallikreins (KLK4-KLK15) have been associated with several types of cancer. For example, hK4, hK5, hK6, hK7, hK8, hK10, hK11, hK13 and hK14 are emerging biomarkers for ovarian, breast, prostate and testicular cancer. New evidence raises the possibility that some kallikreins are directly involved with cancer progression. We here review the evidence linking kallikreins and cancer and their applicability as novel biomarkers for cancer diagnosis and management.     

血清hK2、AMACR联合PSA检测对前列腺癌诊断及预后判断的临床价值

目的:探讨血清腺激肽释放酶2（hK2）、甲基酰基辅酶A消旋酶（AMACR）联合前列腺特异性抗原（PSA）检测对前列腺癌诊断及预后判断的临床价值。方法:前瞻性选取2016年01月至2018年01月收治的前列腺癌患者50例为前列腺癌组和良性前列腺增生患者50例为良性前列腺增生组。并以同期健康查体者50例为对照组。检测比较三组血清hK2、AMACR和PSA水平,Logistic分析血清hK2、AMACR和PSA水平对前列腺癌发生状况的影响,ROC曲线分析血清hK2、AMACR和PSA水平联合检测对前列腺癌的诊断效能。对前列腺癌组进行为期3年的随访,记录患者复发和生存情况。比较不同复发和生存预后情况患者的基线血清hK2、AMACR和PSA水平,Logistic分析基线血清hK2、AMACR和PSA水平对前列腺癌患者复发和生存预后的影响,ROC曲线分析血清hK2、AMACR和PSA水平联合检测对前列腺癌患者复发和死亡早期评估效能。结果:前列腺癌组血清hK2、AMACR水平高于良性前列腺增生组和对照组（P＜0.05）;而良性前列腺增生组和对照组血清hK2、AMACR水平比较差异无统计学意义（P＞0.05）。前列腺癌组、良性前列腺增生组和对照组的血清PSA水平依次降低（P＜0.05）。Logistic分析结果显示,血清hK2、AMACR和PSA水平对前列腺癌发生状况具有明显影响（P＜0.05）。ROC曲线分析结果显示血清hK2、AMACR和PSA水平联合检测对前列腺癌具有良好的诊断效能。前列腺癌组50例患者的3年复发率和生存率分别为56%（28/50）和52%（26/50）。前列腺癌组复发患者的血清hK2、AMACR和PSA水平均高于未复发患者（P＜0.05）;且前列腺癌组死亡患者的血清hK2、AMACR和PSA水平均高于存活患者（P＜0.05）。Logistic分析结果显示,血清hK2、AMACR和PSA水平均受到前列腺癌患者复发和生存预后的影响（P＜0.05）。ROC曲线分析结果显示血清hK2、AMACR和PSA水平联合检测对前列腺癌患者复发和死亡均具有良好的早期评估效能。结论:血清hK2、AMACR和PSA水平在前列腺癌患者中表达水平较高,具有一定的前列腺癌诊断价值,且可能作为预后早期预测的有效参考指标。     

Human kallikrein 7 induces epithelial-mesenchymal transition-like changes in prostate carcinoma cells: a role in prostate cancer invasion and progression

Human tissue kallikrein 7 (hK7), a chymotrypsin-like secreted serine protease, catalyzes the degradation of intercellular adhesive structures in the cornified layer of the skin, leading to desquamation. Thus, hK7 is implicated in cancer invasion and metastasis. Although hK7 is highly expressed in prostate tissues, its biological role in prostate cancer progression is poorly understood. In the current study, we established an hK7-expressing cell model for prostate tumors by stably transfecting prostate carcinoma 22RV1 and DU145 cells with an expression vector encoding hK7. We found that there were no obvious differences in cell proliferation between cells overexpressing hK7 and cells transfected with empty vector (p>0.05). Intriguingly, a Matrigel invasion assay revealed that hK7 remarkably increased the migration and invasion of prostate cancer cells (p<0.01). Furthermore, hK7 induced epithelial-mesenchymal transition-like changes in prostate carcinoma cells, as evidenced by scattered cellular growth, mesenchyma-like morphology, and up-regulated expression of vimentin, a mesenchymal marker. These novel findings suggest that hK7 plays an important role in mediating prostate cancer progression and that hK7 promotes invasion and metastasis, at least in part, through inducing the epithelial-mesenchymal transition of prostatic carcinoma cells.     

Characterization of human kallikreins 6 and 10 in ascites fluid from ovarian cancer patients.

OBJECTIVES: Human kallikreins 6 (hK6) and 10 (hK10) are secreted serine proteases. We previously found that hK6 and hK10 are highly overexpressed in epithelial ovarian tumors and demonstrated that serum levels of hK6 and hK10 are valuable biomarkers for ovarian cancer diagnosis and prognosis. Our aim is to purify and characterize these two kallikreins from ascites fluid of ovarian cancer patients. METHODS: Protein concentrations of hK6 and hK10 in ovarian cancer ascites fluids were measured with ELISA-type immunoassays. hK6 and hK10 were purified from the ascites fluids with immunoaffinity columns, followed by reverse-phase high performance liquid chromatography. Purified hK6 and hK10 were then subjected to N-terminal sequencing. Enzymatic analyses were performed with synthetic fluorogenic peptides. RESULTS: hK6 and hK10 were present in ovarian cancer ascites fluid at concentrations ranging from 0.2-571 and 0.7-220 microg/l, respectively. The majority of hK6 and hK10 in the ascites fluids were present in the free (uncomplexed) form. Both hK6 and hK10 purified from the ascites fluid were zymogens with a molecular mass of 30 kDa. Purified hK6 exhibited trypsin-like enzymatic activity, whereas no enzymatic activity was observed for purified hK10. The enzymatic activity of hK6 could be suppressed by a neutralizing monoclonal antibody. CONCLUSIONS: The majority of hK6 secreted by the ovarian tumor cells into the ascites fluid are present in the uncomplexed, zymogen form, possessing weak trypsin-like enzymatic activity. All hK10 present in ovarian cancer ascites fluids are in the uncomplexed, zymogen form and have no detectable enzymatic activity.     

Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma.

PURPOSE: Human kallikrein 10 (hK10; also known as the normal epithelial cell-specific 1 gene and protein) is a secreted serine protease, which belongs to the human kallikrein family. It has been reported that hK10 is down-regulated in breast and prostate cancer cell lines and that it may function as a tumor suppressor. Recently, we developed a highly sensitive and specific immunoassay for hK10 and found that this protein is abundantly expressed in ovarian tissue. In this study, we measured quantitatively hK10 levels in ovarian cancer cytosolic extracts and evaluated the prognostic value of this biomarker in ovarian cancer. EXPERIMENTAL DESIGN: Specimens from eight normal ovarian tissues, eight ovarian tissues with benign disease, and 182 ovarian tumors were investigated. RESULTS: hK10 concentration in ovarian tumor cytosols ranged from 0 to 84 ng/mg of total protein, with a median of 2.6. This median was highly elevated in comparison with normal and benign ovarian tissues (P < 0.001). A cutoff of 1.35 ng/mg was selected to categorize tumors as hK10 high and hK10 low. With chi(2) test and Fisher's exact test, high concentration hK10 was found to be associated with advanced disease stage, serous histological type, suboptimal debulking, and large residual tumor (>1 cm; all P < 0.05). hK10 status was additionally correlated with clinical outcome, including progression-free (PFS) and overall survival (OS) using the Cox model. In univariate analysis, we found that patients with hK10 high tumors were more likely to die and relapse, in comparison with patients with hK10 low tumors (hazards ratios for PFS and OS were 1.93 and 2.42, respectively; P < 0.05). Although this correlation disappeared after the entire patient population was subjected to multivariate analysis, it remained significant in the subgroup of patients with stage III/IV ovarian cancer (hazards ratios for PFS and OS were 1.98 and 2.12, respectively; P < 0.05). CONCLUSIONS: Our results indicate that hK10 is a new, independent, unfavorable prognostic marker, especially for late-stage ovarian cancer.     

Human kallikrein 2 (hK2), but not prostate-specific antigen (PSA), rapidly complexes with protease inhibitor 6 (PI-6) released from prostate carcinoma cells.

Human kallikrein 2 (hK2) is a secreted, trypsin-like protease that shares 80% amino acid sequence identity with prostate-specific antigen (PSA). hK2 has been shown to be a serum marker for prostate cancer and may also play a role in cancer progression and metastasis. We have previously identified a novel complex between human kallikrein 2 (hK2) and protease inhibitor 6 (PI-6) in prostate cancer tissue. PI-6 is an intracellular serine protease inhibitor with both antitrypsin and antichymotrypsin activity. In the current study we have shown that PI-6 forms a rapid in vitro complex with hK2 but does not complex with PSA. Recombinant mammalian cells expressing both hK2 and PI-6 showed hK2-PI-6 complex in the spent media only after cell death and lysis. Similarly, LNCaP cells expressing endogenous hK2 and PI-6 showed extracellular hK2-PI-6 complex formation concurrently with cell death. Immunostaining of prostate cancer tissues with PI-6 monoclonal antibodies showed a marked preferential staining pattern in cancerous epithelial cells compared with noncancerous tissue. These results indicate that the hK2-PI-6 complex may be a naturally occurring marker of tissue damage and necrosis associated with neoplasia. Both hK2 and PI-6 were shed into the lumen of prostate cancer glands as granular material that appeared to be cellular necrotic debris. The differential staining pattern of PI6 in tissues suggests a complex regulation of PI-6 expression that may play a role in other aspects of neoplastic progression.     

Association of kallikrein expression in nipple aspirate fluid with breast cancer risk

Human kallikreins (hK) 2, 3, 6 and 10 are expressed in breast and prostate tissue. hK2 and hK3 (prostate-specific antigen, PSA) are used to screen for prostate cancer. hK6 and hK10 are downregulated in breast cancer compared to normal breast tissue. We demonstrated that levels of PSA in nipple aspirate fluid (NAF) are lower in women with breast cancer than in normal women. We hypothesize that the expression of hK2, 3, 6 and 10 are related and important in detecting breast cancer. The goals of this study are to determine the level of expression of kallikreins in NAF and serum, the association of hK2, 3, 6 and 10 in NAF, and the association of each of the kallikreins with breast cancer. In NAF from 275 women, hK3, 6 and 10 were detectable in >/= 90% and hK2 in 74% of samples analyzed. NAF levels were highest for hK6 and lowest for hK2, regardless of cancer and menopausal status. hK3 was detectable in 15/29 (52%) and hK2 in 0/29 serum samples collected from 6 women. hK2 and hK3 were concentrated in NAF vs. matched serum. The 4 kallikreins were associated with the exception of hK2 with hK6 or hK10. PSA levels were higher in normal pre- than postmenopausal subjects (but not women with breast cancer), whereas levels of hK2, 6 and 10 did not differ by menopausal status. hK2 and PSA were associated with both pre- and postmenopausal breast cancer; hK6 and 10 were not. hK2 and PSA were more associated with pre- than postmenopausal breast cancer. Using logistic regression, PSA and menopausal status provided the best model of breast cancer prediction, with a sensitivity of 91% and specificity of 39%. In conclusion, 4 kallikreins are expressed in NAF. hK2 and PSA, and hK6 and hK10 are highly associated. Higher premenopausal PSA levels suggest the influence of ovarian steroids. PSA shows the most promise in aiding in the early detection of breast cancer.     

The serum concentration of human kallikrein 10 represents a novel biomarker for ovarian cancer diagnosis and prognosis

Human kallikrein 10 (hK10) is a secreted serine protease that is highly expressed in ovarian tissue. We hypothesized that hK10 might represent a novel serological marker for ovarian cancer. We quantified by immunoassay, hK10 in sera from 97 normal women (controls), 141 patients with benign gynecologic diseases, and 146 patients with ovarian cancer. We then examined the diagnostic and prognostic value of this measurement in ovarian cancer. We found that normal serum hK10 ranged from 50 to 1040 ng/liter (mean = 439 ng/liter). hK10 concentration is significantly elevated in serum of presurgical ovarian cancer patients (range: 106-11,746 ng/liter; mean = 1067 ng/liter) but not in serum of patients with benign gynecologic diseases (range: 120-1200 ng/liter; mean = 447 ng/liter). When a cutoff of 700 ng/liter was selected (diagnostic specificity = 90%), the diagnostic sensitivity for ovarian cancer is 54%. About 35% of CA125-negative ovarian cancer patients (CA125 < 23 kU/liter) were hK10 positive at 90% specificity. In patients with stage I/II ovarian cancer, use of these two markers in combination results in a 21% increase in sensitivity, at 90% specificity, compared with CA125 alone. High serum hK10 was strongly associated with serous epithelial type, late-stage, advanced grade, large residual tumor (>1 cm), suboptimal debulking, and no response to chemotherapy (all Ps < 0.001). In univariate Cox survival analysis, high serum hK10 is associated with increased risk for relapse and death (hazard ratio = 2.59 and 3.15, respectively, P 相似文献   

Human kallikrein 13 protein in ovarian cancer cytosols: a new favorable prognostic marker.

PURPOSE: Human kallikrein 13 (hK13; encoded by the KLK13 gene) is a secreted serine protease expressed in endocrine tissues, including the prostate, testis, breast, and ovary. We have previously reported steroid hormone regulation of the KLK13 gene and its clinical value as a marker of favorable prognosis in breast cancer at the mRNA level. We hypothesized that hK13 may represent a potential biomarker for ovarian carcinomas. PATIENTS AND METHODS: Using a newly developed enzyme-linked immunosorbent assay (ELISA), hK13 levels were quantified in 131 ovarian tumor extracts and correlated with various clinicopathological variables and outcome (progression-free survival [PFS], overall survival [OS]), over a median follow-up period of 42 months. RESULTS: hK13 concentration in ovarian tumor cytosols ranged from 0 to 18.4 ng/mg of total protein. An optimal cutoff value of 0.13 ng/mg (67(th) percentile) was selected, based on the ability of hK13 values to predict the PFS of the study population, to categorize tumors as hK13-positive or negative. Women with hK13-positive tumors most often had early stage (stage I/II) disease, no residual tumor after surgery and optimal debulking success (P <.05). Univariate and multivariate Cox regression analyses revealed that patients with hK13-positive tumors had a significantly longer PFS and OS than hK13-negative patients (P <.05). Kaplan-Meier survival curves further confirmed a reduced risk of relapse and death in women with hK13-positive tumors (P =.007 and P =.002, respectively). CONCLUSION: These results indicate that hK13 is an independent marker of favorable prognosis in ovarian cancer.     

Human kallikrein 8 protein is a favorable prognostic marker in ovarian cancer.

Human kallikrein 8 (hK8/neuropsin/ovasin; encoded by KLK8) is a steroid hormone-regulated secreted serine protease differentially expressed in ovarian carcinoma. KLK8 mRNA levels are associated with a favorable patient prognosis and hK8 protein levels are elevated in the sera of 62% ovarian cancer patients, suggesting that KLK8/hK8 is a prospective biomarker. Given the above, the aim of the present study was to determine if tissue hK8 bears any prognostic significance in ovarian cancer. Using a newly developed ELISA, hK8 was quantified in 136 ovarian tumor extracts and correlated with clinicopathologic variables and outcome [progression-free survival (PFS); overall survival (OS)] over a median follow-up period of 42 months. hK8 levels in ovarian tumor cytosols ranged from 0 to 478 ng/mg total protein, with a median of 30 ng/mg. An optimal cutoff value of 25.8 ng/mg total protein (74th percentile) was selected based on the ability of hK8 values to predict the PFS of the study population and to categorize tumors as hK8 positive or negative. Women with hK8-positive tumors most often had lower-grade tumors (G1), no residual tumor after surgery, and optimal debulking success (P < 0.05). Univariate and multivariate analyses revealed that patients with hK8-positive tumors had a significantly longer PFS and OS than hK8-negative patients (P < 0.05). Kaplan-Meier survival curves further confirmed a reduced risk of relapse and death in women with hK8-positive tumors (P = 0.001 and P = 0.014, respectively). These results indicate that hK8 is an independent marker of favorable prognosis in ovarian cancer.     

Downregulation of human kallikrein 10 (KLK10/NES1) by CpG island hypermethylation in breast, ovarian and prostate cancers.

OBJECTIVE: The human kallikrein 10 (KLK10)/normal epithelial cell-specific-1 (NES1) gene is highly expressed in normal mammary, ovary and prostate cells, but its expression is dramatically decreased in cancer cell lines. Recently, it has been shown that CpG island hypermethylation of the KLK10 gene is responsible for the tumor-specific loss of KLK10 gene expression in certain breast cancer cell lines. METHOD: We examined the role of CpG island hypermethylation in the tumor-specific loss of KLK10 expression in breast, ovarian and prostate cancers. We treated cells with the demethylating agent 5-aza-2'-deoxycytidine (dC) and monitored changes in KLK10 mRNA by RT-PCR and secreted hK10 protein expression by ELISA. The following cell lines were used: MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-1, T-47D and BT-474 (breast); BG-1, MDAH-2774, HTB-75, HTB-161, PA-1 and ES-2 (ovary), and LNCaP and PC-3 (prostate). RESULTS: Upregulation of KLK10 mRNA levels, which was accompanied by an increase in secreted hK10 protein concentration, was observed for a subset of breast, ovarian, and prostate tumor cell lines after 5-aza-2'-dC. Genomic sequencing of sodium-bisulfite-treated DNA demonstrated that CpG sites within the KLK10 gene exon 3 were highly methylated. Hypermethylation of exon 3 CpG regions was also detected in primary ovarian cancers. CONCLUSION: These data suggest that CpG island hypermethylation plays an important role in the downregulation of kallikrein 10 mRNA and protein expression, but it cannot explain the pattern of expression of this gene in all cell lines or tissue tested.