Polycomb protein EZH2 suppresses apoptosis by silencing the proapoptotic miR-31.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and suppresses gene expression by catalyzing histone H3 methylation on lysine 27. EZH2 is overexpressed in metastatic prostate cancer and has been shown to promote cell proliferation and metastasis. Here we show that EZH2 also suppresses prostate cancer apoptosis by coordinating the epigenetic silencing of two proapoptotic microRNAs (miRNA), miR-205 and miR-31. We previously reported that miR-205 promotes apoptosis by targeting antiapoptotic protein Bcl-w and miR-205 is silenced in prostate cancer through promoter methylation. In this study, we found that EZH2 suppresses miR-31 expression by trimethylation of lysine 27 on histone 3 on the miR-31 promoter. SiRNA knockdown of EZH2 increased miR-31 expression and decreased the antiapoptotic protein E2F6 (E2F transcription factor 6) (a target of miR-31), resulting in the sensitization of prostate cancer cells to docetaxel-induced apoptosis. Conversely, overexpression of EZH2 blocked docetaxel-induced apoptosis. We further demonstrated that miR-205 silencing is linked to miR-31 silencing through EZH2. Suppression of miR-205 with an miRNA inhibitor caused an increase of EZH2 protein, which in turn inhibited miR-31 expression. Conversely, overexpression of miR-205 decreased EZH2 protein and increased miR-31 expression. In paired human prostate cancer specimens and adjacent normal tissues, we observed that the decrease of miR-205 expression correlated with EZH2 overexpression and miR-31 silencing. Thus, EZH2 integrates the epigenetic silencing of miR-205 and miR-31 to confer resistance to chemotherapy-induced apoptosis.

Use of tumor dynamics to clarify the observed variability among biochemical recurrence nomograms for prostate cancer.

BACKGROUND: Nomograms for biochemical recurrence (BCR) of prostate cancer (PC) after radical prostatectomy can yield very different prognoses for individual patients. Since the nomograms are optimized on different cohorts, the variations may be due to differences in patient risk-factor distributions. In addition, the nomograms assign different relative scores to the same PC risk factors and rarely stratify for tumor growth rate. METHODS: We compared BCR-free probabilities from the GPSM model with a cell kinetics (CK) model that uses the individual's tumor state and growth rate. We first created a cohort of 143 patients that reproduced the GPSM patient distribution in Gleason score, Prostate specific antigen (PSA), Seminal vesicle involvement and Margin status since they form the GPSM score. We then performed 143 CK calculations to determine BCR-free probabilities for comparison with the GPSM results for all scores and with four other prominent nomograms for a high-risk patient. RESULTS: The BCR-free probabilities from the CK model agree within 10% with those from the GPSM study for all scores once the CK model parameters are stratified in terms of the GPSM risk factors and the PSA doubling time (PSADT). However, the probabilities from widely used nomograms vary significantly. CONCLUSIONS: The CK model reproduces the observed GPSM BCR-free probabilities with a broad stratification of model parameters for PC risk factors and can thus be used to describe PC progression for individual patients. The analysis suggests that nomograms should stratify for PSADT to be predictive.

FOXOs, cancer and regulation of apoptosis.

Forkhead box O (FOXO) transcription factors are involved in multiple signaling pathways and play critical roles in a number of physiological and pathological processes including cancer. The importance of FOXO factors ascribes them under multiple levels of regulation including phosphorylation, acetylation/deacetylation, ubiquitination and protein-protein interactions. As FOXO factors play a pivotal role in cell fate decision, mounting evidence suggests that FOXO factors function as tumor suppressors in a variety of cancers. FOXOs are actively involved in promoting apoptosis in a mitochondria-independent and -dependent manner by inducing the expression of death receptor ligands, including Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand, and Bcl-2 family members, such as Bim, bNIP3 and Bcl-X(L), respectively. An understanding of FOXO proteins and their biology will provide new opportunities for developing more effective therapeutic approaches to treat cancer.

The androgen receptor: a potential target for therapy of prostate cancer.

The androgen receptor plays a pivotal role in the prostate. Its primary function is to provide responsive gene products for differentiation and growth, but under abnormal conditions it contributes to the development of prostate cancer. The goal of this review is to elucidate the molecular functions of the androgen receptor and its role in prostate cancer. Initially the function of the androgen receptor will be described. Next, the clinical diagnosis, epidemiological impact, and treatments of androgen-dependent and -independent prostate cancer will be discussed. Finally we will examine how the mechanism of androgen action has played a role in the translation of new therapies and how this may influence future treatment modalities of prostate cancer.

Retinoic acid-induced stimulation of sodium iodide symporter expression and cytotoxicity of radioiodine in prostate cancer cells.

We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.

The role of PTEN in the progression and survival of prostate cancer.

PTEN (phosphatase and tensin homologue deleted on chromosome-10), a dual specificity phosphatase, is a tumor suppressor gene whose inactivation has been associated with many different types of cancer including prostate cancer. Prostate adenocarcinoma is one of the most commonly diagnosed malignancies afflicting the male population in both the United States and Europe. The frequency of PTEN inactivation appears to increase during the progression of prostatic cancer. The physical loss of the PTEN genetic locus in prostate cancer progression has been well characterized, however the molecular implication of this loss of PTEN remains enigmatic. The purpose of this review is to describe the functional role of PTEN in the molecular pathogenesis of prostatic disease. We review the function of PTEN discussing its association with the phosphoinositol 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) signal transduction pathways. Additionally, we discuss the role of PTEN in the regulation of apoptotic pathways involving the anti-apoptotic gene bcl-2 and the pro-apoptotic ligand TRAIL. We also review the mechanisms that can lead to the loss of PTEN function. We describe genetic inactivation including loss of heterozygosity, haploinsufficiency and mutation. We conclude by outlining epigenetic loss including methylation, post-translational modifications and oxidative stress.

In vivo sodium iodide symporter gene therapy of prostate cancer.

Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of thyroid-specific expression of the sodium iodide symporter (NIS). Here we explore the efficacy of a novel form of gene therapy using adenovirus-mediated in vivo NIS gene transfer followed by (131)I administration for treatment of prostate cancer. Prostate cancer xenografts in nude mice injected with an adenovirus carrying the NIS gene linked to the cytomegalovirus (CMV) promoter revealed highly active uptake of radioiodine. Following administration of 3 mCi of (131)I, we observed an average tumor volume reduction of 84 +/- 12%. These results show for the first time that in vivo NIS gene delivery into non-thyroidal tumors is capable of inducing accumulation of therapeutically effective radioiodine doses and might therefore represent an effective and potentially curative therapy for prostate cancer.

Androgen receptor signaling in androgen-refractory prostate cancer.

Prostate cancer is the second most prevalent cancer in males in the United States. Standard therapy relies on removing, or blocking the actions of, androgens. In most cases, this therapy results in a regression of the cancer because the prostate and most primary prostate tumors depend on androgens for growth and the avoidance of apoptosis. However, a portion of the cancers eventually relapse, at which point they are termed "androgen refractory" and can no longer be cured by conventional therapy of any type. The precise molecular events that lead from androgen-sensitive prostate cancer to androgen-refractory prostate cancer are, therefore, of great interest. This review seeks to identify specific molecular events that may be linked directly to the progression to androgen-refractory cancer. Some of the mechanisms appear to involve the androgen receptor (AR) directly and include mutations in, or amplification of, the AR gene in a manner that allows the AR to respond to low doses of androgens, other steroids, or antiandrogens. In a less direct manner, coactivators may increase the sensitivity of the AR to androgens and even other nonandrogenic substances through a number of mechanisms. Additional indirect mechanisms that do not result from mutation of the AR may involve activation of the AR by peptide growth factors or cytokines or may involve bypassing the AR entirely via other cellular pathways. Identification of the role of these mechanisms in the progression to androgen-refractory prostate cancer is critical for developing therapies capable of curing this disease.

Tyrphostin AG825 triggers p38 mitogen-activated protein kinase-dependent apoptosis in androgen-independent prostate cancer cells C4 and C4-2.

Prostate cancer (PCa) progression is aided by abnormal autocrine growth factor loops. We screened for small cell-permeable inhibitors of receptor tyrosine kinases that could block their signaling and trigger cell death in PCa cell lines. We found that the human epidermal growth factor receptor (HER)-2/neu inhibitor tyrphostin AG825 is preferentially toxic to PCa cells that are phenotypically androgen independent. These effects were dose and time dependent in the human LNCaP, C4, and C4-2 cell line models of progression and correlated with the inhibition of HER-2/neu phosphoactivation and its down-regulation. In addition, we show that the inhibition of HER-2/neu signaling with AG825 triggers an imbalance between extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, which leads to p38-dependent apoptosis. Inhibition of HER-1 with Compound 56 had no effect. These findings suggest that the androgen-independent C4 and C4-2 cells can be killed by selectively inhibiting their HER-2/neu signaling pathway and provide insights into the mechanism of action of AG825 in PCa cells.

Role of PI3K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state.

The mechanisms by which prostate cancer (PCa) cells progress to a hormone refractory state are poorly understood. The progression process under androgen ablation conditions involves the survival of at least a portion of malignant cells and their eventual proliferation in an androgen-independent manner. The goal of this study was to investigate the role of PI3K signaling in such a progression. Using an in vitro model of androgen ablation, we show that after removal of androgen support, the human PCa cell line LNCaP initially arrested in G(1) and trans-differentiated into neuroendocrine-like cells that eventually resumed androgen-independent proliferation. Both acute and chronic androgen ablation resulted in an increase in basal levels of PI3K and Akt activity, which were sustained throughout the progression process. Under these conditions, inhibition of PI3K, pharmacologically or with ectopic expression of PTEN, arrested cell proliferation and blocked progression to the androgen-independent state. In contrast, LNCaP cells in the presence of androgens were marginally sensitive to PI3K inhibition. During the chronic stage of androgen deprivation, androgen-independent proliferation correlated with diminished p27(kip1) protein levels, whereas PI3K and Akt activity remained elevated. At this stage, PI3K inhibition rapidly triggered accumulation of p27(kip1), cell cycle arrest, and cell death. PI3K modulated p27(kip1) levels at least in part by regulating its rate of degradation. Taken together, these data show that androgen ablation alone can increase PI3K-Akt activation, which supports survival after acute androgen ablation and proliferation during chronic androgen deprivation. Successful progression to the androgen-independent state in the LNCaP cell line model requires intact PI3K signaling.

PTEN induces chemosensitivity in PTEN-mutated prostate cancer cells by suppression of Bcl-2 expression.

The tumor suppressor gene PTEN (MMAC1/TEP1) is lost frequently in advanced prostate cancer (PCa). However, the function of PTEN in tumorigenesis is not understood fully. In this study, we demonstrate that expression of Bcl-2 in prostate tumors correlates with loss of the PTEN protein. This finding was verified by studies in the PCa cell lines DU145, PC-3, LNCaP, and an androgen-refractory subline of LNCaP. Transient transfection of PTEN into the PTEN-null cells resulted in decreased levels of Bcl-2 mRNA and protein. These effects appear to be mediated at the level of gene transcription, since a Bcl-2 promoter-reporter construct was down-regulated by ectopic expression of PTEN in LNCaP cells. The inhibition of Bcl-2 required the lipid-phosphatase activity of PTEN and was blocked by overexpression of a constitutively active form of Akt. Moreover, the transcription-regulatory protein cAMP-response element-binding protein (CREB) may be involved, since decreased phosphorylation of CREB at Ser(133) was detected following PTEN expression, and ectopic expression of CREB repressed completely the PTEN-induced inhibition of Bcl-2 promoter activity. Furthermore, cotransfection of Bcl-2 and PTEN expression vectors rescued PTEN-induced cell death but not G(1) cell cycle arrest. Finally, forced expression of PTEN sensitized LNCaP cells to cell death induced by staurosporine, doxorubicin, and vincristine, and this chemosensitivity was attenuated by exogenous expression of Bcl-2. Taken together, these data demonstrate that loss of PTEN leads to up-regulation of the bcl-2 gene, thus contributing to survival and chemoresistance of PCa cells. These findings suggest that the PTEN gene and its regulated pathway are potential therapeutic targets in prostate cancer.

State of research for prostate cancer: Excerpt from the report of the Prostate Cancer Progress Review Group.

The following is an excerpt from a report entitled: "Defeating Prostate Cancer: Crucial Directions for Research." The report, which was submitted to the Advisory Committee to the Director of the National Cancer Institute, summarizes the findings and recommendations of the Progress Review Group.

Robust prostate-specific expression for targeted gene therapy based on the human kallikrein 2 promoter.

Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.

Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter.

Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.

Androgen receptors in prostate and skeletal muscle.

Regulatory control of the androgen receptor has been studied most extensively in prostatic tissue. Additional information has been obtained from the study of androgen receptors in pituitary and hypothalamic tissues, and these results can be extrapolated to regulatory mechanisms in both prostate and skeletal muscle. The present work provides a brief summary of studies on androgen action on muscle, gives an overview of mechanisms of androgen action on target tissues, and focuses on several aspects of regulation of androgen receptor (AR) gene expression. These include some characteristics of AR gene transcription start sites; the role of the cyclic adenosine monophosphate, protein kinase C, and protein kinase A pathways in mediating androgen receptor function; and tissue-variable regulatory elements inhibiting AR gene expression.

Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2.

15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) is a highly specific activator of the peroxisome proliferator-activated receptor gamma (PPAR-gamma). We investigated the effect of 15d-PGJ2 on three human prostate cancer cell lines, LNCaP, DU145, and PC-3. Western blotting demonstrated that PPAR-gamma1 is expressed predominantly in untreated prostate cancer cells. Treatment with 15d-PGJ2 caused an increase in the expression of PPAR-gamma2, whereas PPAR-gamma1 remained at basal levels. PPARs alpha and beta were not detected in these cells. Lack of lipid accumulation, increase in CCAAT/enhancer binding proteins (C/EBPs), or expression of aP2 mRNA indicated that adipocytic differentiation is not induced in these cells by 15d-PGJ2. 15d-PGJ2 and other PPAR-gamma activators induced cell death in all three cell lines at concentrations as low as 2.5 microM (similar to the Kd of PPAR-gamma for this ligand), coinciding with an accumulation of cells in the S-phase of the cell cycle. Activators for PPAR-alpha and beta did not induce cell death. Staining with trypan blue and propidium iodide suggested that, although the plasma membrane appears intact by electron microscopy, disturbances are evident as early as 2 h after treatment. Mitochondrial transmembrane potentials are significantly reduced by 15d-PGJ2 treatment. In addition, treatment with 15d-PGJ2 resulted in cytoplasmic changes, which are indicative of type 2 (autophagic), nonapoptotic programmed cell death.

Expression of a prostate-associated protein, human glandular kallikrein (hK2), in breast tumours and in normal breast secretions.

The recent demonstration of human glandular kallikrein (hK2) expression in a breast carcinoma cell line has suggested that this putatively prostate-restricted, steroid hormone-regulated protease may also be expressed in breast epithelium in vivo and secreted into the mammary duct system. Given that the only substrate yet identified for hK2 activity is the precursor of prostate-specific antigen (PSA), the expression of which in breast carcinomas may be associated with favourable prognosis, our purpose was to examine the expression pattern of both hK2 and PSA in breast tumour tissues. Cytosolic extracts of 336 primary breast carcinomas prepared for routine oestrogen receptor (ER) and progesterone receptor (PR) analysis, as well as 31 nipple aspirates from six women with non-diseased mammary glands, were assayed for hK2 and PSA using immunofluorometric assays developed by the authors. In the tumour extracts, measurable hK2 and PSA concentrations were detected in 53% and 73% of cases respectively, and were positively correlated to each other (r = 0.59, P = 0.0001). Higher concentrations of PSA and hK2 were found in tumours expressing steroid hormone receptors (P = 0.0001 for PSA and P = 0.0001 for hK2, by Wilcoxon tests for both ER and PR), and both PSA (r = 0.25, P = 0.0001) and hK2 (r = 0.22, P = 0.0001) correlated directly with PR levels. A negative correlation between patient age and PSA (r = -0.12, P = 0.03) was also found. Both proteins were present in nipple aspirate fluid at relatively high concentrations which were positively correlated (r = 0.53, P = 0.002). The molecular weights of the immunoreactive species quantified by the hK2 and PSA assays were established by high-performance liquid chromatography (HPLC) and were consistent with the known molecular weights of hK2 and PSA. Together these data provide the first evidence, to our knowledge, that both malignant breast tissue and normal breast secretion contain measurable quantities of hK2, and that the degree of hK2 expression or secretion is directly proportional to the expression of PSA and steroid hormone receptors. hK2 expression may therefore be a marker of steroid hormone action in breast tissue.

Detection of metastatic prostate cancer using a splice variant-specific reverse transcriptase-polymerase chain reaction assay for human glandular kallikrein.

We developed a highly sensitive splice variant-specific reverse transcriptase-PCR (RT-PCR) assay for human glandular kallikrein (hK2) mRNA and tested its ability to detect metastatic disease in men with clinically localized prostate cancer. An RT-PCR assay using primers spanning intron IV and including a significant portion of the 3' untranslated region of the hKLK2 gene, with maximum nonhomology to both hK1 and hK3, was developed. The limit of detection of the assay was five copies of hK2 cDNA and one LNCaP cell in 10(9) lymphoblasts. RT-PCR-hK2 was performed on preoperative peripheral blood specimens from 228 consecutive radical prostatectomy patients as well as 7 metastatic prostate cancer patients and 14 healthy men without prostate cancer. This new RT-PCR-hK2 assay amplifies two distinct fragments. The larger fragment (hK2-U) is approximately 680 bp in length and corresponds to the amplified product of a previously reported splice variant in the splice donor site of intron IV in the hKLK2 gene. The smaller fragment (hK2-L) is approximately 643 bp in length and corresponds to the amplified product of the native hK2 mRNA. Whereas the RT-PCR-hK2-L assay was positive in 71% of our patients with metastatic prostate cancer, 14% of healthy control men also tested positive. By univariate (P = 0.028) and multivariate (P = 0.0269) analysis, which controlled for preoperative PSA, clinical stage, and biopsy Gleason score, RT-PCR-hK2-L status added prognostic information to the prediction of lymph node-positive disease. We have developed a new RT-PCR assay which demonstrates a high sensitivity for detecting hK2 mRNA. Preoperative RT-PCR-hK2-L status helps predict pathological lymph node positivity in patients with clinically localized prostate cancer.

Differential expression and allelotyping of the p73 gene in neuroblastoma.

p73 has recently been identified as a candidate imprinted tumor suppressor gene in neuroblastoma. To determine the possible involvement of this gene in the pathogenesis of neuroblastoma, we analyzed allelic expression, screened for mutations and determined MYCN copy numbers in 31 primary neuroblastoma tumor samples. Interestingly, the gene was biallelically expressed in 50% (4/8) of informative neuroblastomas, which suggests that activation of the normally silenced allele of this gene plays an important role in the tumorigenesis of neuroblastoma. However, no tumor-specific mutations were identified although 15 polymorphisms were detected in this gene. We also detected a very strong association between a C91T polymorphism and MYCN copy number in this tumor. The T allele was detected in 8/17 (47%) neuroblastomas without MYCN amplifications but not detected in cases with MYCN amplifications (0/14). The biological significance of this association, however, is unknown. Overall the data suggest that p73 may play an important role in the pathogenesis of neuroblastoma but that the true tumor suppressor gene localized to this area still remains to be identified.

Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis.

OBJECTIVES: Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha2-macroglobulin and alpha1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. METHODS: Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritech's Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). RESULTS: The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. CONCLUSIONS: In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.