15-deoxy-Delta(12,14)-prostaglandin J2 inhibits G2-M phase progression in human breast cancer cells via the down-regulation of cyclin B1 and survivin expression.

The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a growth inhibitory effect on cancer cells, and this effect is linked to the induction of apoptosis or cell cycle arrest. Induction of apoptosis by 15d-PGJ(2) is associated with the down-regulation of anti-apoptotic proteins. G(0)-G(1)-->S phase progression is inhibited by 15d-PGJ(2) via the degradation of cyclin D1. In this study, we further investigated the mechanism by which 15d-PGJ(2) inhibits cancer cell growth by using the breast cancer cell lines MCF-7 and T-47D. Treatment with 20 microM 15d-PGJ(2) for 72 h completely blocked the growth in both cell lines. However, the proportions of apoptotic MCF-7 and T-47D cells were 21.1% and 40.9%, respectively, indicating that the induction of apoptosis did not appear to fully account for growth inhibition by 15d-PGJ(2). Cell cycle analysis using cells synchronized at the G(0)-G(1) or S phase revealed that 15d-PGJ(2) blocked not only G(0)-G(1)-->S phase progression but also G(2)-M phase progression. The expression of both cyclins D1 and B1 was decreased by 15d-PGJ(2). Furthermore, 15d-PGJ(2) inhibited aurora-B kinase activity, which coincided with the down-regulation of survivin. Thus, 15d-PGJ(2) induced cell cycle arrest at the G(2)-M phase via inhibition of cyclin B1 expression and aurora-B kinase activity. We conclude that survivin may be an important target for 15d-PGJ(2), and its down-regulation may lead to a decrease in aurora-B kinase activity.

Development of a monoclonal antibody against Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) that can detect EBNA-LP expressed in P3HR1 cells.

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) fused to glutathione-S-transferase. The antibody detected endogenous and exogenous EBNA-LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA-LP. While another monoclonal antibody to EBNA-LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA-LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA-LP, especially investigations into the phenotypes of mutant EBNA-LP expressed in P3HR1 cells.

Mutations of the p53 gene and loss of heterozygosity at chromosome 17p13.1 are associated with increased survivin expression in breast cancer.

Expression of survivin, a member of the inhibitor-of-apoptosis (IAP) family, is elevated in fetal tissues and in various human cancers originating in the breast, lung, prostate, colon, pancreas, and stomach. Since overexpression of the survivin gene has been linked to poor patient survival in several cancers, survivin may be an important prognostic marker. Mechanisms up-regulating survivin gene expression in cancer are poorly understood. Recently, wild-type p53 was found to repress expression of the survivin gene by binding to the survivin promoter, thereby inhibiting promoter activity. Further, loss of heterozygosity (LOH) at 17p13 distal to the p53 gene is associated with more aggressive behavior of breast cancers. We therefore tested the hypothesis that not only p53 gene mutation but also LOH at 17p13 can up-regulate survivin expression in breast cancer. Survivin mRNA expression was greater in cancers than in uninvolved tissues (p < 0.0001). Mutations of the p53 gene were detected in 5 of 25 tumors; higher survivin gene expression was evident in these. LOH at the D17S938 locus (17p13.1) was found in 10 of 25 tumors, and most of these also showed increased survivin gene expression. Thus expression of survivin may be regulated not only by p53 but additionally by a putative tumor suppressor gene located at 17p13 distal to the p53 gene.

PCD1, a novel gene containing PDZ and LIM domains, is overexpressed in human breast cancer and linked to lymph node metastasis.

BACKGROUND: Despite surgical removal of the primary tumor of breast cancer in patients with apparently localized disease, relapse at local or distant sites may occur because undetectable micrometastases were present at the time of diagnosis. Identification of molecules associated with breast cancer metastasis suggests possible new treatments. PCD1, a gene encoding a new member of the PDZ and LIM domain-containing protein family, recently was identified. We examined the relationships between PCD1 mRNA expression in breast cancers and metastasis. MATERIALS AND METHODS: PCD1 mRNA expression in breast cancer tissues was examined using a quantitative reverse transcription polymerase chain reaction. RESULTS: PCD1 mRNA expression was greater in cancers than in noncancerous tissues (p < 0.0001). In addition, high PCD1 gene expression was more frequent in patients with lymph node metastasis. CONCLUSION: PCD1 appears to contribute to breast cancer progression and nodal metastasis, thus representing a potential target molecule in breast cancer diagnosis and treatment.

A novel aspartic protease gene, ALP56, is up-regulated in human breast cancer independently from the cathepsin D gene.

Tumor cell invasion requires expression of degradative enzymes such as plasminogen activator, collagenase, and cathepsins. Cathepsin D, a lysosomal aspartic protease produced constitutively in human breast cancer cell lines, also has mitogenic activity in breast cancer cells. Additionally, high cathepsin D expression is associated with increased risk of metastasis in patients with node-negative breast cancer. Recently, a novel aspartic protease gene, ALP56 (aspartic-like protease 56kDa), has been identified. To examine possible interrelationships we quantitated ALP56 mRNA and cathepsin D mRNA in breast cancers using reverse transcription polymerase chain reaction. ALP56 mRNA expression was greater in cancers than in noncancerous tissues (p < 0.0001), as was expression of cathepsin D mRNA. ALP56 gene expression was dose-dependently down-regulated in T-47D breast cancer cells treated with estradiol, while cathepsin D was up-regulated. Expression of ALP56 mRNA in estrogen receptor (ER)-positive breast cancers was less than that in ER-negative cancers, and mRNA expression for ALP56 and cathepsin D did not correlate with one another. Thus ALP56 as well as cathepsin D may be a useful target molecule in breast cancer treatment.

Overexpression of early growth response-1 as a metastasis-regulatory factor in gastric cancer.

BACKGROUND: To investigate the potential role of a nuclear transcription factor, early growth response-1 (Egr-1), in formation and progression of gastric cancer, we compared its expression in gastric cancers with that in non-cancerous tissues. MATERIALS AND METHODS: Egr-1 mRNA expression was measured using TaqMan RT-PCR. The corresponding protein expression was examined immunohistochemically. RESULTS: Egr-1 mRNA expression was significantly higher in gastric cancer tissues than in normal mucosa (p < 0.0005). These differences were also reflected by protein product expression. Moreover, Egr-1 mRNA expression was higher in cases with metastasis to lymph nodes or remote organs. In cultured gastric cancer cells known to have a high metastatic potential, expression of this mRNA was higher than that of parental cells. CONCLUSION: It was suggested that Egr-1 has a significant role in carcinogenesis and in cancer progression, especially metastasis. Measurement of this mRNA should be useful for evaluation of the metastatic potential of gastric cancer.

Enhanced expression of the UROC28 gene in human breast cancer: relationship to ERBB2 gene expression.

BACKGROUND: ERBB2, a highly important oncogene in invasive breast cancer, is not only a prognostic factor but also a predictive marker for response to therapeutic agents. Recently, An et al. (10) identified a novel gene, UROC28, that is also overexpressed in breast cancer. To examine possible interrelationships, we quantitated UROC28 and ERBB2 mRNA in breast cancers. MATERIALS AND METHODS: The expression of UROC28 and ERBB2 mRNA in breast cancer tissues were determined using RT-PCR. The expression was also examined in T-47D breast cancer cells treated with estrogen. RESULTS: UROC28 mRNA expression was greater in cancers than in noncancerous tissues (p < 0.0001), as was ERBB2 mRNA. ERBB2 and UROC28 gene expression was dose-dependently down-regulated in T-47D breast cancer cells treated with estradiol. However UROC28 mRNA and ERBB2 mRNA expression did not correlate with one another. CONCLUSION: These results indicate that UROC28 may be a useful target molecule in breast cancer diagnosis and treatment, complementing ERBB2.

Selection of an internal control gene for quantitation of mRNA in colonic tissues.

BACKGROUND: GAPDH, beta-actin and 18S rRNA are widely employed as internal control genes, with the assumption that they are expressed constitutively to similar degrees in different cells and tissues and under different experimental conditions. In this study, we tested this assumption by assessment of the transcription of these three genes in human colonic tissues using a quantitative RT-PCR. RESULTS: GAPDH transcription was significantly greater in both colonic adenomas and cancers than in normal mucosa. In addition, transcription of beta-actin was significantly increased in cancers. The expression of 18S rRNA was essentially constant among these various tissues. Stable expression of 18S rRNA was observed during the growth of colonic cancer cells stimulated with serum, but both GAPDH and beta-actin transcription were up-regulated, coinciding with cell proliferation. CONCLUSION: These results indicate that 18S rRNA is more reliable than GAPDH and beta-actin as an internal control gene for quantitative comparison of mRNA in colonic cancers.