[Variation of urinary PCA3 following transrectal ultrasound-guided prostate biopsy]. / Variation du PCA3 urinaire après biopsie de prostate transrectale échoguidée.

INTRODUCTION: Serum PSA is known to rise slightly following an attentive digital rectal examination (DRE) and dramatically following prostatic biopsy. The aim of this study was to evaluate the PCA3 response in these situations. PATIENTS AND METHODS: In 15 consecutive men undergoing transrectal ultrasound-guided needle biopsy of the prostate and who gave their informed consent, urinary PCA3 was determined twice: at a first consultation, urine being sampled immediately after an attentive DRE and second within 2 hours after the biopsy. The mean interval between the two samplings was 14 days (median 15). PCA3 measurements were centralized and performed by the same biologist. At least twelve cores were taken using a biopsy gun with an 18-gauge needle. Changes in PCA3 levels were studied. RESULTS: Mean age of the 15 men was 67.3 years (range 50.9-79.1). Mean (median) pre-biopsy total and %free PSA were respectively 6.6 ng/ml (5.7) and 15.8% (15.5). Mean prostate volume was 43.6 cm(3). Seven patients complained of mild LUTS. DRE was suspicious in eight patients. Of the 15 men, 6 (40%) had adenocarcinoma on biopsy (all clinically confined to the prostate). Median (range) Gleason score was 6 (6-7). Median PCA3 score (range) before and after prostatic biopsy were respectively 36 (9-287) and 27 (5-287) with no significant difference between the two groups (sign test for matched series p > 0.05). The median variation between pre- and post-biopsy PCA3 was -18%. When considering a PCA3 cut-off of 35, two patients changed group: one patient had 51 before and 31 after (PSA 4.6; no cancer on prostate biopsy) and the second had 36 before and 27 after (PSA 5.6; low-risk PCa). The figure represents the PCA3 values for each case (squares for the pre-biopsy and diamonds for the post-biopsy). When considering only the six patients with PCA, median (mean) PCA3 score before and after prostatic biopsy were respectively 51.5 (60.8) and 44.5 (54.8) with no significant difference between the two groups (sign test for matched series p > 0.5) and a median variation between pre- and post-biopsy PCA3 of 1.5%. CONCLUSIONS: Prostate biopsy did not alter significantly urinary PCA3 value. This confirms what was theoretically expected.

Prenatal diagnosis of common aneuploidies using multiplex quantitative fluorescent polymerase chain reaction.

OBJECTIVE: Prenatal diagnosis of foetal trisomies is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures and it is expensive. Here, we report a retrospective study of quantitative fluorescent polymerase chain reaction (QF-PCR) for prenatal detection of trisomies 13, 18 and 21. METHODS: QF-PCR was performed on a total of 447 amniotic fluids blindly analysed without any knowledge of the cytogenetic results and 43 samples with known karyotype. All samples were tested with at least 4 small tandem repeat markers specific for each chromosome 13, 18 or 21. RESULTS: QF-PCR results on amniotic fluid were consistent with conventional cytogenetic data. QF-PCR detected 5 cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13 and 1 case with Klinefelter's syndrome. CONCLUSIONS: QF-PCR has proved to be very useful in clinical settings, since it allows the detection of major numerical disorders in a few hours after sampling and thus reduces parental anxiety.

A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ.

A number of Ags recognized by tumor-reactive T cells have been characterized, including nonmutated gene products and a variety of epitopes shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with an HLA-B7-restricted renal cell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphocytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV complementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (ORF) (a +1 frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence-based peptide binding assay using transfected T2 cells. Constitutive expression of this alternative ORF protein was observed in all transformed HLA-B7+ renal cell lines that were recognized in cytotoxicity assays by the TILs. The intestinal carboxyl esterase gene is transcribed in renal cell carcinoma tumors as well as in normal liver, intestinal, or renal tissues. Mutation of the natural ATG translation initiation site did not alter recognition, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three AUG codons that may be used as alternative translation initiation sites in the +1 ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of the alternative ORF. These results further extend the types of Ags that can be recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion).

Characterization of two monoclonal antibodies against the RON tyrosine kinase receptor.

RON is a receptor protein tyrosine kinase belonging to the hepatocyte growth factor (HGF) receptor family. Using Récepteur d'Origine Nantais (RON) transfected cell lines, Macrophage Stimulating Protein (MSP) was identified as the ligand of RON. RON is synthesized as a single chain precursor, which subsequently is cleaved to yield a disulfide-linked heterodimer, with a 40-kDa alpha chain and a 150-kDa beta chain. Activation of RON by MSP results in cell migration, shape change, and proliferation. The present work centers on the production and characterization of two monoclonal antibodies (MAbs) to RON called ID-1 and ID-2. Antibodies were generated by immunization of mice with Madin-Darby Canine Kidney (MDCK) cells expressing human RON (clone RE7). Both antibodies recognized the mature and precursor form of RON. The specificity of the anti-RON antibodies was confirmed using a hepatocarcinoma cell line HepG2 expressing both task MET and RON receptors. Specific immunoprecipitation with ID-1 and ID-2 or anti-MET antibody followed by Western blotting under reducing conditions with rabbit polyclonal antibodies against RON and MET showed that our anti-RON antibodies recognize specifically the RON receptor. Ligand binding experiments showed that both antibodies are able to block the binding of radiolabeled MSP to RON and showed also that the antibodies recognize two different epitopes in the molecule. The blocking of MSP binding to RON by the anti-RON antibodies was confirmed by inhibition of cell migration induced by MSP in HT-29-D4 cells. Significant immunostaining was not observed in any subpopulation of whole blood with either ID-1 or ID-2. We analyzed the expression of RON receptor in a number of human hematopoietic and nonhematopoietic cells lines by flow cytometry. We found a strong mean of fluorescence intensity (MFI) in colon adenocarcinoma cells SW620 and HT-29-D4, low MFI in SVK14 and HepG2 cells, and no immunostaining in melanoma, lymphoma, and leukemia cells. Immunohistochemistry revealed that RON was expressed in germinal centers of tonsil, in skin, small intestine, and colon. These antibodies defined RON as CDw136 during the last leucocyte typing VI.

Macrophage-stimulating protein activates Ras by both activation and translocation of SOS nucleotide exchange factor.

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2, but not Shc, associated with the activated RON-beta chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Ras might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.

Structure of the promoter for the human macrophage stimulating protein receptor gene.

The macrophage stimulating protein receptor is a receptor protein tyrosine kinase of the met/hepatocyte growth factor receptor family. Binding of the macrophage stimulating protein to its receptor provokes changes in cell morphology and motility. Here we report the structure of the promoter of the gene coding for this receptor. The major transcription start sites have been identified. The 5' flanking region has characteristics of other receptor tyrosine kinase gene promoters, namely several GC boxes but the absence of a TATA box. Deletion analysis shows that multiple elements are needed for full promoter activity.

Identification of the ron gene product as the receptor for the human macrophage stimulating protein.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

A novel putative receptor protein tyrosine kinase of the met family.

By successive screenings of cDNA libraries prepared from human tumours and from human foreskin keratinocytes, we have isolated overlapping cDNAs coding for a novel protein which we call Ron, with sequence characteristics of a receptor protein tyrosine kinase. Ron is a 1400 amino acid protein structurally similar to the 1408 amino acid product of the C-MET proto-oncogene, the receptor for hepatocyte growth factor and scatter factor. The two proteins have 63% overall sequence identity in their intracellular regions. We have localised the RON gene to human chromosome region 3p21, a region frequently deleted in small cell carcinoma of the lung and in renal cell carcinoma, and which is believed to harbour unidentified tumour suppressor genes. Interestingly, normal lung tissue contains transcripts of the RON gene.

Activation of transcription via AP-1 or CREB regulatory sites is blocked by protein tyrosine phosphatases.

Transcriptional activation endowed by AP-1 or CREB binding sites can be significantly reduced in transient transfection tests by expression from the corresponding cloned cDNAs of protein tyrosine phosphatases. Both the protein tyrosine phosphatase 1B and the T-cell protein tyrosine phosphatase, as well as a novel form of this latter protein generated by an alternative splicing even show this activity. The effect is specific, as none of the protein tyrosine phosphatases alters transcriptional activation by either the estrogen receptor, GAL4, or a GAL4-VP16 fusion protein. Furthermore, the activities of the SV40 early gene promoter and a Moloney murine leukemia virus long terminal repeat promoter are not reduced by these phosphatases. We conclude that a yet to be identified protein phosphorylated on tyrosine is necessary for a full transcriptional response via AP-1 or CREB binding sites.

Multiple mRNAs code for proteins related to the BEK fibroblast growth factor receptor.

The BEK transmembrane protein tyrosine kinase is a receptor for both acidic and basic fibroblast growth factors. We identify several different transcripts which code for BEK-related proteins. These proteins differ from BEK in regions expected to control receptor activity. Thus, some of the proteins have altered extracellular, ligand-binding domains, and others an altered carboxy-terminal tail. Still other forms of BEK differ only in their juxtamembrane domains. Sequencing of parts of the BEK gene shows that alternative splicing of the premessenger can account for at least some of this diversity. In particular, an apparently tissue specific, mutually exclusive splicing of two internal exons permits both the previously described K-SAM mRNA and the BEK mRNA to be derived from the same premessenger.