Xavier Farré

Expression of cathepsins B and S in the progression of prostate carcinoma

Cathepsins B and S (CatB, CatS) are lysosomal cysteine proteases which, among other functions, appear to play a role in cancer progression in different tumor models due to their matrix‐degrading properties. To investigate their possible involvement in the development of prostate carcinoma, we immunohistochemically analyzed CatB and CatS in 38 primary human prostatic adenocarcinomas, as well as concomitant high‐grade prostatic intra‐epithelial neoplasia, nodular hyperplasia and normal tissue. CatB expression was observed in 28 (74%) and CatS in 32 (84%) carcinomas, being concomitant in 24 cases (63%). High‐grade intra‐epithelial neoplasia expressed CatB in 20/23 cases (87%), and a similar result was obtained for CatS, with expression of both coinciding in 18 cases (78%). In non‐neoplastic tissue, strong expression of both proteases was observed in macrophages, inflamed glands and transitional metaplasia, whereas atrophic glands and basal cells of normal glands displayed intense CatB positivity. We conclude that CatB and CatS are often expressed together in neoplastic prostatic cells from pre‐invasive to invasive and clinically detectable stages, suggesting a putative role in local invasion, though other functions cannot be ruled out.

EXPRESSION OF THE NUCLEOSIDE-DERIVED DRUG TRANSPORTERS hCNT1, hENT1 AND hENT2 IN GYNECOLOGIC TUMORS

Deoxynucleoside analogs are used in the treatment of a variety of solid tumors. Their transport across the plasma membrane may determine their cytotoxicity and thus nucleoside transporter (NT) expression patterns may be of clinical relevance. Lack of appropriate antibodies for use in paraffin‐embedded biopsies has been a bottleneck to undertake high‐throughput analysis of NT expression in solid tumors. Here we report the characterization of 2 new antibodies raised against the low‐affinity equilibrative NTs, hENT1 and hENT2, suitable for that purpose. These 2 antisera, along with a previously characterized antibody that specifically recognizes the high‐affinity Na‐dependent concentrative NT, hCNT1, have been used to analyze, using a tissue array approach, NT expression in gynecologic cancers (90 ovarian, 80 endometrial and 118 uterine cervix carcinomas). Human CNT1 was not detected in 33% and 39% of the ovarian and uterine cervix carcinomas, respectively, whereas hENT1 and hENT2 expression was significantly retained in a high percentage of tumors (91% and 96% for hENT1, 84% and 98% for hENT2, in ovarian and cervix carcinomas, respectively). Only a few endometrial carcinomas (15%) were found to be negative for hCNT1, but they all retained hENT1 and hENT2 expression. In ovarian cancer, the loss of all 3 NT proteins was a more common event in the clear cell histologic subtype than in the serous, mucinous and endometrioid histotypes. In uterine cervix tumors, the loss of expression of hCNT1 was significantly associated with the adenocarcinoma subtype. In summary, hCNT1 was by far the isoform whose expression was most frequently reduced or lost in the 3 types of gynecologic tumors analyzed. Moreover, NT expression is related to the type of gynecologic tumor and its specific subtype, hCNT1 protein loss being highly correlated with poor prognosis histotypes. Since hCNT1, hENT1 and hENT2 recognize fluoropyrimidines as substrates, but with different affinities, this study anticipates high variability in drug uptake efficiency in solid tumors.

Expression of p27/kip1 is down-regulated in human prostate carcinoma progression

p27Kip1 is a cyclin‐dependent kinase inhibitor whose down‐regulation has been observed in several tumour models, including breast, colorectal, and gastric carcinomas. The purpose of this study was to assess p27Kip1 protein expression in normal and benign prostatic epithelia as well as the possible existence of abnormalities in prostate carcinoma progression. p27Kip1 expression was immunohistochemically analysed in 51 normal tissue samples, 11 nodular hyperplasias (NH), 22 high‐grade prostatic intraepithelial neoplasias (PIN), 56 localized prostate adenocarcinomas, and 19 metastases. Immunoblotting was performed in ten cases. Normal prostate epithelium and NH showed diffuse and intense p27Kip1 nuclear expression in most cases. A significant p27Kip1 down‐regulation was observed in many carcinomas when compared with benign epithelium. Forty‐seven cases (84 per cent) were low p27Kip1 expressors (<50 per cent positive cells) and nine cases (16 per cent) were high p27Kip1 expressors. p27Kip1 down‐regulation was also consistently seen in PIN. Fourteen out of 19 metastases (74 per cent) were low p27Kip1 expressors. Six metastatic samples had their corresponding primary tumour analysed and three cases showed decreased expression in the metastasis. It is concluded that p27Kip1 is constitutively expressed in normal and benign prostatic tissue. This expression is clearly down‐regulated in neoplastic progression from the preinvasive lesions through invasive carcinoma and metastases and this therefore occurs in early stages of neoplastic transformation. Copyright

PTOV-1, a Novel Protein Overexpressed in Prostate Cancer, Shuttles between the Cytoplasm and the Nucleus and Promotes Entry into the S Phase of the Cell Division Cycle

PTOV1 was recently identified as a novel gene and protein during a differential display screening for genes overexpressed in prostate cancer. The PTOV1 protein consists of two novel protein domains arranged in tandem, without significant similarities to known protein motifs. By immunohistochemical analysis, we have found that PTOV1 is overexpressed in 71% of 38 prostate carcinomas and in 80% of samples with prostate intraepithelial neoplasia. High levels of PTOV1 in tumors correlated significantly with proliferative index, as assessed by Ki67 immunoreactivity, and associated with a nuclear localization of the protein, suggesting a functional relationship between PTOV1 overexpression, proliferative status, and nuclear localization. In quiescent cultured prostate tumor cells, PTOV1 localized to the cytoplasm, being excluded from nuclei. After serum stimulation, PTOV1 partially translocated to the nucleus at the beginning of the S phase. At the end of mitosis, PTOV1 exited the nucleus. Transient transfection of chimeric green fluorescent protein-PTOV1 forced the entry of cells into the S phase of the cell cycle, as shown by double fluorescent imaging for green fluorescent protein and for Ki67, and also by flow cytometry. This was accompanied by greatly increased levels of cyclin D1 protein in the transfected cells. These observations suggest that overexpression of PTOV1 can contribute to the proliferative status of prostate tumor cells and thus to their biological behavior.

Alterations of cell cycle-regulatory genes in prostate cancer.

Deregulated proliferation is one of the main events in neoplastic transformation, and this has prompted increased attention being given to the understanding of the mechanisms involved in cell cycle regulation and its alterations. The ‘retinoblastoma pathway’, a key effector controlling G1-S phase transition, includes several oncogenes and tumour suppressor genes which display a wide range of abnormalities with potential usefulness as markers of evolution or treatment response in prostate cancer. Among these, the existence of p53 mutations seems to predict resistance to radiotherapy or systemic treatment, and p16 overexpression or p27 downregulation seems to serve as markers of poor evolution. The well-established existence of a critical hormonal role in prostate carcinogenesis coupled with the relationship of androgenic activity and regulation of several cell cycle modulators forces cell cycle control in the prostate to be envisioned as a highly complex steroid-influenced system, which will undoubtedly have critical implications in the future management of prostate cancer patients.

Tissue macroarrays (mIcrochops) for gene expression analysis

Abstract. We describe a simple system of tissue arraying with multiple tissue fragments obtained with a biopsy punch from selected areas of paraffin blocks. The new blocks thus constructed allow multiple tissue sections in which the uniform shape of the fragments coupled with a geometrical display and a significant amount of tissue per case allows a dependable, cost-effective way to screen tumors or other kinds of tissues with techniques such as immunohistochemistry. This system avoids the disadvantages of previous laborious methods of tissue arraying, such as expensive equipment and scarce tissue sampling, and it can be implemented in any institution with minimal cost and elaboration.

Expression of Ep‐CAM, but not of E48, associates with nodal involvement in advanced squamous cell carcinomas of the larynx

To evaluate epithelial cell adhesion molecule (Ep‐CAM) and E48 expression, and their relationship with histological differentiation and nodal metastasis, in laryngeal squamous cell carcinomas (SCC).