Prostate-specific membrane antigen (PSMA) enzyme activity is elevated in prostate cancer cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a glutamate carboxypeptidase that cleaves terminal carboxy glutamates from both the neuronal dipeptide N-acetylaspartylglutamate (NAAG) and gamma-linked folate polyglutamate. The prostate enzyme has activity in both the membrane and cytosolic fractions termed PSMA and PSMA', respectively. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we quantitated the enzymatic activity of PSMA and PSMA' in normal, benign prostatic hyperplasia (BPH), and prostate cancer (PC) tissues from radical prostatectomies. PSMA enzyme activity was evaluated in each tissue type and expressed per milligram protein and epithelial cell content. RESULTS: PSMA and PSMA' enzyme activities were significantly elevated in prostate cancer when compared to normal prostate tissue and BPH. Ratios of PSMA to PSMA' were also decreased in BPH as compared to cancerous and normal tissue. CONCLUSIONS: Prostate carcinogenesis is associated with an elevation in PSMA and PSMA' enzyme activity. In contrast, no such enhancement in PSMA activity is observed with benign neoplastic changes in BPH. Thus, the enhancement observed in prostate cancer is not simply related to a generalized prostatic hyperplasia, but is specific to its malignancy.

Suramin potently inhibits the enzymatic activity of PSM.

BACKGROUND: The polysulfonated napthlyurea suramin has shown significant antitumor activity in patients with hormone-refractory metastatic prostate cancer. The mechanism by which suramin exerts this effect is unknown. In 1993, prostate-specific membrane antigen (PSM) was identified as a prostate biomarker that is elevated in hormone-refractory and metastatic prostate cancer. PSM is a glutamate exocarboxypeptidase capable of cleaving the terminal alpha-linked glutamate from the dipeptide N-acetyl-aspartyl-glutamate (NAAG) and the gamma-linked glutamates from folate polyglutamate. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we tested whether suramin had any effect on the enzymatic activity of PSM. RESULTS: We demonstrate that suramin potently inhibits the enzymatic activity of PSM with a K(i) = 15 nM and 68 nM for the membrane-associated and soluble forms of PSM, respectively. In addition, we show that suramin inhibition of PSM enzyme activity displays the kinetics of a classic competitive inhibitor. CONCLUSIONS: This is one of the most potent activities described for suramin to date and may represent a portion of its pharmacologic and/or toxicological mechanism of action.

Characterization of the enzymatic activity of PSM: comparison with brain NAALADase.

BACKGROUND: The prostate cancer marker prostate-specific membrane antigen (PSM) is highly homologous to the brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is known to cleave terminal carboxy glutamates from both the neuronal peptide N-acetylaspartylglutamate (NAAG) and folate polyglutamate. In this report, we compare the NAAG hydrolyzing activity of NAALADase and the prostate enzyme PSM. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we compared the pharmacological and kinetic properties of the brain and prostate enzymes. RESULTS: Eight normal prostate tissues from different species exhibited NAAG hydrolyzing activity. Among 14 cancer cell lines examined, activity was observed in human LNCaP, PC-82, and rat Dunning G and AT-1 cells. Brain exhibited membrane-localized activity exclusively, while the prostate enzyme had activity in both membrane and cytosolic fractions. The only observed pharmacological difference was the sensitivity to their putative substrates, folate polyglutamate and NAAG. Kinetically, the soluble form of the prostate enzyme had two catalytic sites, while the membrane-bound form exhibited single site kinetics with a lower Vmax than the brain enzyme, which may suggest a less active hydrolase in the prostate. CONCLUSIONS: The brain enzyme NAALADase and the prostate enzyme PSM are remarkably similar. The importance of the differences in substrate specificities and kinetic parameters remains to be elucidated.

Enhanced efficacy of a novel controlled release paclitaxel formulation (PACLIMER delivery system) for local-regional therapy of lung cancer tumor nodules in mice.

The efficacy of systemic chemotherapy for non-small cell lung cancer (NSCLC) has improved with newer agents. However, the response rates and prolonged survival times achieved by chemotherapy remain modest, and these small gains are obtained at the cost of significant toxicity. In this study, the efficacy of a controlled release formulation of paclitaxel was compared with conventional paclitaxel in animals with human lung cancer xenografts. Paclitaxel (10%) was encapsulated in a proprietary polymer in the form of microspheres (PACLIMER Delivery System). Tumor nodules comprised of two different cell lines (A549 and H1299) were treated by a single i.p. or intratumoral administration of conventionally formulated paclitaxel or a single intratumoral injection of the PACLIMER Delivery System. In vitro testing demonstrated that paclitaxel was released slowly from the microspheres with >80% released after 90 days. Direct comparison of the highest dose for all formulations (24 mg/kg) showed that for nodules comprised of either NSCLC cell line, growth of the PACLIMER Delivery System-treated nodules were inhibited significantly more than the groups treated with conventional paclitaxel or the vehicle controls. Tumor volume doubling times for A549 and H1299 nodules treated with PACLIMER Delivery System were 60 and 35 days, respectively, compared with 10 and 11 days, respectively, in the nodules treated with the conventional paclitaxel by intratumoral administration. We conclude that intratumoral administration of the PACLIMER Delivery System may substantially increase the efficacy of paclitaxel for the therapy of local-regional NSCLC.

Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression.

Progesterone receptor (PR) is an estrogen-stimulated gene which has a CpG island that is heavily methylated in a significant fraction of estrogen receptor (ER)-negative/PR-negative human breast cancers and cell lines, including MDA-MB-231 cells. Treatment of MDA-MB-231 cells with the demethylating agent, 5-aza-2'-deoxycytidine (deoxyC) led to demethylation and expression of ER and PR. However, simultaneous treatment with antiestrogen prevented PR transcription, suggesting that demethylation of PR alone is not sufficient to reactivate the PR gene. To examine the effects of ER on the methylation status of the PR CpG island, we stably transfected MDA-MB-231 cells with an inducible expression vector for ER. Surprisingly, in two cell clones, we found that induction of PR gene expression by ligand-bound ER does not require demethylation of the PR CpG island. In contrast, induction of PR transcription was inhibited by blocking the interaction of ER with SRC-1A, a coactivator of ER function. For the first time, we show that a transcription factor with the potential to remodel heterochromatin can activate gene expression without altering the methylation status of the CpG island. These results raise the possibility that demethylation and histone acetylation are distinct but complementary mechanisms for destabilizing heterochromatin and activating transcription.

Mapping of ER gene CpG island methylation-specific polymerase chain reaction.

Southern analysis has shown that DNA from 25% of primary estrogen receptor (ER) alpha-negative breast tumors displays aberrant methylation at one site within the ER gene CpG island. To examine more sites and increase sensitivity, we developed a methylation-specific PCR assay to map methylation of the entire ER CpG island. The island was unmethylated in normal breast tissue and ER-positive breast cancer cell lines, but extensively methylated in all ER-negative cell lines and breast tumors examined. In addition, some of the ER-positive/progesterone receptor-negative and ER-positive/progesterone receptor-positive tumors (about 70% and 35%, respectively) displayed methylation of the ER CpG island, suggesting that this heterogeneity within tumor cell populations could potentially shed light on the etiology of ER-negative recurrent tumors arising from ER-positive tumors.

The loss of estrogen and progesterone receptor gene expression in human breast cancer.

Hormone responsiveness is a critical determinant of breast cancer progression and management, and the response to endocrine therapy is highly correlated with the estrogen receptor (ER)3 and progesterone receptor (PR) status of tumor cells. Thus, key areas of study in breast cancer are those mechanisms that regulate ER and PR expression in normal and malignant breast tissues. One-third of all breast cancers lack ER and PR; these conditions are associated with less differentiated tumors and poorer clinical outcome. In addition, approximately one-half of ER-positive tumors lack PR protein and patients with this phenotype are less likely to respond to hormonal therapies than those whose tumors express both receptors. Since PR is induced by ER; its presence is a marker of a functional ER. In this review, we will discuss possible mechanisms for loss of ER and PR gene expression, especially structural changes within each gene including deletions, polymorphisms or methylation. Improved understanding of the pathways that lead to loss of ER and/or PR proteins should allow the development of better predictive indicators as well as novel therapeutic approaches to target these hormone-independent cancers.

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors.

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.

E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas.

Expression of the Ca(2+)-dependent, homotypic cell:cell adhesion molecule, E-cadherin (E-cad), suppresses tumor cell invasion and metastasis in experimental tumor models. Decreased E-cad expression is common in poorly differentiated, advanced-stage carcinomas. These data implicate E-cad as an "invasion suppressor" gene. The mechanism by which E-cad is silenced in advanced stage carcinomas is unclear. In this report, we show that: (a) the 5' CpG island of E-cad is densely methylated in E-cad-negative breast and prostate carcinoma cell lines and primary breast carcinoma tissue but is unmethylated in normal breast tissue; (b) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, partially restores E-cad RNA and protein levels in E-cad-negative breast and prostate carcinoma cell lines; and (c) and E-cad promoter/CAT construct is expressed in both E-cad-positive and -negative breast and prostate carcinoma cell lines, indicating that these cells have the active transcriptional machinery necessary for E-cad expression. Our data demonstrate that frequent loss of E-cad expression in human breast and prostate carcinomas results from hypermethylation of the E-cad promoter region.

Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers.

The tumor suppressor gene CDKN2/p16/MTS1, located on chromosome 9p21, is frequently inactivated in many human cancers through homozygous deletion. Recently, we have reported another pathway of inactivation that involves loss of transcription associated with de novo methylation of a 5' CpG island of CDKN2/p16 in lung cancers, gliomas, and head and neck squamous cell carcinomas. We now show that this aberrant CpG island methylation also occurs frequently in cell lines of breast cancer (33%), prostate cancer (60%), renal cancer (23%), and colon cancer (92%) and is associated with loss of transcription. Primary tumors of the breast (31%) and colon (40%) also displayed de novo methylation of this CpG island. This alteration of p16 in colon cancer was particularly striking, since inactivation does not occur through homozygous deletion in this tumor type. Our data show that in tumors, de novo methylation of the 5' CpG island is a frequent mode of inactivation of CDKN2/p16 and also firmly demonstrate that CDKN2/p16 is one of the most frequently altered genes in human neoplasia.

Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression.

Approximately one third of breast cancers grow independently of estrogen, lack detectable estrogen receptor (ER) protein, and rarely respond to hormonal treatment. Previous studies correlated the lack of ER gene expression in ER-negative breast tumor cells with hypermethylation of a CpG island in the 5' region of the ER gene. In order to determine whether demethylation of the ER gene in the ER-negative human breast cancer cell line MDA-MB-231 could affect ER transcription, cells were treated with two inhibitors of DNA methylation, 5-azacytidine or 5-aza-2'-deoxycytidine. DNA from cells treated with either drug became partially demethylated at several methylation-sensitive restriction enzyme sites, including HhaI, NotI, and SacII, within the ER CpG island. This demethylation correlated with reexpression of the ER gene as detected by reverse transcriptase-PCR and production of ER protein as detected by Western blot analysis. ER produced in drug-treated cells was functionally active as demonstrated by its ability to activate transcription of estrogen-responsive genes. These results suggest that DNA methylation of the ER CpG island may play a role in suppression of ER gene expression in ER-negative breast cancer cells.

The mitochondrial permeability transition. Interactions of spermine, ADP, and inorganic phosphate.

Mitochondria that have accumulated Ca2+ can be induced to undergo a permeability transition: the inner membrane becomes nonselectively permeable to small (< 1500 daltons) solutes. Our laboratory has recently identified the polyamine spermine as an inhibitor of the permeability transition of isolated rat heart and liver mitochondria. Here, we have used swelling of liver mitochondria as an indicator of transition occurrence to investigate the connection between spermine, another transition antagonist, ADP, and several key triggering agents: P(i), Ca2+, and t-butyl hydroperoxide (t-BH). Our results demonstrate that: 1) ADP strongly inhibits only the swelling induced by P(i); transitions induced by t-BH and Ca2+ are minimally affected. 2) The sensitivity of the permeability transition to P(i) is enhanced in mitochondria depleted of adenine nucleotides. 3) Incubation with P(i) decreases mitochondrial ADP and ATP content. 4) Spermine inhibits less well in adenine nucleotide-depleted than control mitochondria, regardless of triggering agent. 5) Spermine and ADP act synergistically to inhibit the transition. 6) ADP replenishment makes P(i) a worse triggering agent. Triggering by Ca2+ and t-BH is enhanced. 7) P(i) overcomes spermine inhibition; Ca2+ and t-BH do not. We propose that P(i) triggers the transition by lowering the matrix concentration of the inhibitor ADP and that spermine inhibits the transition by enhancing ADP effectiveness. In addition, these data clearly distinguish the triggering action of P(i) from that of Ca2+ and t-BH.

Spermine inhibition of the permeability transition of isolated rat liver mitochondria: an investigation of mechanism.

Mitochondria that have accumulated Ca2+ can be induced to undergo a permeability transition: the inner membrane becomes nonselectively permeable to small (< 1500 Da) solutes. The molecular mechanism(s) underlying this transition, which is Ca(2+)-dependent and cyclosporin A-sensitive, has yet to be clearly elucidated. Our laboratory has recently identified the polyamine spermine as an inhibitor of the permeability transition of isolated rat heart mitochondria. In this study, we have used spermine, in combination with a series of triggering agents, to clarify several mechanistic details of the transition process in isolated rat liver mitochondria. Mitochondrial swelling was monitored as an indicator of transition occurrence. Our results indicate that: (1) spermine inhibits the permeability transition of isolated rat liver mitochondria; (2) the sensitivity of the permeability transition of liver mitochondria to spermine is highly dependent on the ionic composition of the assay medium; (3) K+ interacts with a site outside the mitochondria to decrease spermine effectiveness; (4) spermine likewise acts at an external site; and (5) the Ca2+ uniporter in its inactive form is not the protein responsible for mediating the permeability transition.

Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria.

The effect of spermine on the permeability transition of the inner mitochondrial membrane of isolated rat heart mitochondria was evaluated. The permeability transition was triggered using a series of agents (t-butyl hydroperoxide, phenylarsine oxide, carboxyatractylate, and elevated Ca2+ and inorganic phosphate concentrations), and was monitored via Ca(2+)-release, mitochondrial swelling and pyridine nucleotide oxidation. By all three criteria, spermine inhibited the transition. A C50 of 0.38 +/- 0.06 (SD) mM was measured for inhibition.