Expression analysis of delta-catenin and prostate-specific membrane antigen: Their potential as diagnostic markers for prostate cancer

The current approach to prostate cancer diagnosis has major limitations including the inability of prostate-specific antigen (PSA) assays to accurately differentiate between prostate cancer and benign prostate hyperplasia (BPH) and the imprecision of transrectal ultrasound (TRUS) biopsy sampling. We have employed cDNA microarray screening to compare gene expression patterns in BPH and tumour samples to identify expression markers that may be useful in discriminating between these conditions. Screening of 3 individual cDNA arrays identified 8 genes with expression 3-fold greater in 6 tumour tissues than in 1 nontumour sample and I BPH sample. Real-time PCR was used to confirm the overexpression of these 8 genes and 12 genes selected from the literature against a panel of 17 tumours and I 1 BPH samples. Two genes, delta-catenin (delta-catenin; CTNND2) and prostate-specific membrane antigen (PSMA; FOLH1), were significantly overexpressed in prostate cancer compared to BPH. Prostate epithelial cells stained positively for S-catenin and PSMA in our prostate cancer tissues, whereas the majority of our BPH tissues were negative for both markers. Thus we have identified delta-catenin (not previously associated with prostatic adenocarcinoma) and confirmed the potential of PSMA as potential candidates for the diagnosis and management of prostate cancer. (C) 2002 Wiley-Liss. Inc.

Expression of MUC1 splice variants in benign and malignant ovarian tumours

MUC1 is expressed on the surface of ovarian cancer cells. Nine different splice variants of MUC1 have been described, but no study has reported on the expression of MUC1 iso-forms in human ovarian cancer. Our study compares patterns of expression of MUC1 splice variants of malignant and benign ovarian tumours. Ovarian tissue samples were taken from patients with benign ovarian tumours (n = 34) and from patients who had surgery for primary (n = 47) or recurrent (n = 8) ovarian cancer. RT-PCR for MUC1 splice variants A, B, C, D, X, Y, Z, REP and SEC was performed and their expression compared to clinical and histopathologic parameters. Variants A, D, X, Y and Z were more frequently expressed in malignant than in benign tumours. All primary ovarian cancer cases were positive for variant REP but negative for variant SEC. No significant association of the expression of MUC1 splice variants with the response to chemotherapy or patient survival could be demonstrated. Expression of MUC1 splice variants A, D, X, Y, Z and REP is associated with the presence of malignancy, whereas expression of MUC1/SEC is associated with the absence of malignancy. (C) 2002 Wiley-Liss, Inc.

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.

Immunity to asexual blood stage malaria and vaccine approaches

The development of a malaria vaccine seems to be a definite possibility despite the fact that even individuals with a life time of endemic exposure do not develop sterile immunity. An effective malaria vaccine would be invaluable in preventing malaria-associated deaths in endemic areas, especially amongst children less than 5 years of age and pregnant women. This review discusses our current understanding of immunity against the asexual blood stage of malaria - the stage that is responsible for the symptoms of the disease - and approaches to the design of an asexual blood stage vaccine.

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.

Therapeutic LMP1 polyepitope vaccine for EBV-assolciated Hodgkin disease and nasopharyngeal carcinoma

Development of an epitope-based vaccination strategy designed to enhance Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) is increasingly being considered as a preferred approach for the treatment of EBV-associated relapsed Hodgkin disease (HD) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane proteins, LMP1 and LMP2, are the only target antigens available for therapeutic augmentation of CTL responses in patients with HD and NPC. Here, we describe preclinical studies using a recombinant poxvirus vaccine that encodes a polyepitope protein comprising 6 HLA A2-restricted epitopes derived from LMP1. Human cells infected with this recombinant polyepitope construct were efficiently recognized by LM1-specific CTL lines from HLAA2 healthy individuals. Furthermore, immunization of HLrA A2/K-b mice with this polyepitope vaccine consistently generated strong LMP1 -specific CTL responses to 5 of the. 6 epitopes, which were readily detected by both ex vivo and in vitro assays. More important, this polyepitope vaccine successfully reversed the outgrowth of LMP1-expressing tumors in HLA A2/Kb mice. These studies provide an important platform for the development of an LMP-based polyepitope vaccine as an immunotherapeutic tool for the treatment of EBV-associated HD and NPC. (C) 2003 by The American Society of Hematology.

E2F modulates keratinocyte squamous differentiation. Implications for E2F inhibition in squamous cell carcinoma

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 ( E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation- insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl- phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.