Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging?

Intensification using peripheral blood stem cells collected after chemotherapy followed by growth factors is being increasingly investigated as an alternative to conventional chemotherapy for mantle cell non-Hodgkin lymphoma. We investigated 14 grades III-IV, t(11;14)-positive cases for contamination of PBSC collected after a polychemotherapy regimen followed by G-CSF. Patients were first treated with a polychemotherapy regimen. There were four CR, seven PR, two refractory and one early death. Seven patients have been transplanted, in whom PBSC were mobilized, using either cyclophosphamide/VP16 or Dexa-BEAM followed by G-CSF. For all patients, whether actually autografted or not, PB cells were tested at the time of regeneration on G-CSF after the first polychemotherapy or after the mobilizing regimen. PCR evaluation of contamination was performed first by a semi-quantitative approach, using serial dilutions of initial DNA, then confirmed using a limiting-dilution analysis. Two patients were not informative (one early death and one without an available molecular marker). PB cells collected at regeneration contained at least one log more lymphoma cells than steady-state blood or marrow, apart from in two cases. Moreover, where a mobilizing treatment diminished tumor burden in the patient, at the same time it increased PB contamination in most cases. We conclude that advanced mantle cell NHL appears to be largely resistant to significant in vivo purging by conventional chemotherapy. Where treatment brings benefits by reducing tumor load, it may at the same time negate it by mobilizing malignant cells into the collections used to intensify. Although the clonogenic potential of this massive infiltration is unknown (only gene marking studies could provide a definitive answer regarding the source of relapses), strategies aimed at reducing the level of contamination in the graft should be considered when designing future protocols.

Introduction of a normal retinoblastoma (Rb) gene into Rb-deficient lymphoblastoid cells delays tumorigenicity in immunodefective mice.

Inactivation of the Rb susceptibility gene occurs in various human cancers and has been associated with tumorigenicity. Rb gene is inactivated in 30% of acute leukaemias. The effect of Rb protein expression was assessed in the lymphoblastoid cell line IM-9 defective for Rb protein, after stable transfection with a wild-type Rb gene. The Rb transgene was under the control of the MoMuLv-LTR. Protein expression by the transduced cells was confirmed by Western blot and flow cytometry analyses. Compared to the parental cell line, growth rate remained unchanged in the Rb transfected clones. In SCID mice however, tumor formation originating from these clones was delayed. The current data suggest therefore that, in this Rb-defective haematopoietic cell line, Rb expression correlates with reduced tumorigenicity but not with reduced growth rate.

Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: implications for the detection of hematogenous prostate cells and standardization.

Circulating prostate cells can be detected in cancer patients by using reverse transcriptase-PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.

Beta-thalassaemia in indigenous Belgians: an update.

Beta-thalassaemia, a widespread autosomal recessive disorder, occurs sporadically in Northern and Western European countries. Molecular analysis of the beta-globin gene has been carried out in 30 members of 15 unrelated indigenous Belgian families which presented with non sideropenic hypochromic and microcytic anaemia. For all of them, extensive search failed to find an ancestor at risk for the disease. The beta-globin genes were first screened for frequent beta-thalassemic mutations by dot-blot hybridization with specific radiolabeled oligonucleotide probes. Direct automated fluorescence-based DNA sequencing and, in one case, Southern blotting were also used. All the 30 patients were found to be heterozygous for a beta-thalassemic mutation. Eight different mutations were identified. Among these, four are commonly found in the Mediterraneans: codon 8 (-AA), IVS-I-1 (G-->A), IVS-1-6 (T-->C) and codon 39 (C-->T); three have occasionally been reported: initiation codon (T-->C) and codon 35 (C-->A) and a rare deletion of 12.6 kb which removes all the beta-globin gene and its flanking regions. A new mutation, a -CC deletion at codon 38/39 was found in one family. These results both at the biological and molecular level show that beta-thalassaemia exist in indigenous Belgian families with no known ancestor a risk for the disease. They also show that clinicians and biologists should keep in mind the existence of beta-thalassaemia in indigenous Belgian families when investigating hypochromic and microcytic anaemia in patients whom the past familial history does not evocate a risk for the disease.

An anti-HIV-1 gag protein rat monoclonal antibody library.

A set of 18 rat monoclonal antibodies (MAbs) directed against human immunodeficiency virus type 1 (HIV-1) gag proteins was derived from 4 independent fusion protocols. The epitopes recognized were delineated using a random fragment expression library representing the whole HIV-1IIIB genome. This panel of rat MAbs was used to analyze the antigenicities of the HIV-1 CAp24 major core protein and the HIV-1 NCp7 nucleocapsid protein. As a result, a limited set of antigenic domains as defined, 3 on CAp24 between amino acids (aa) 195 and 268, 323 and 329, 329 and 352, and one on NCp7 (aa 382-392). Only 4 mouse anti-CAp24 MAbs appeared to recognize the COOH-terminal domain (aa 329-352) defined by the majority of our MAbs. The rat anti-CAp24 (Q1B10) and the rat anti-NCp7 (I5B11) MAbs described here, defined two newly described epitopes, aa 323-329 and aa 382-392.

Isolation of a C-type retrovirus from an HIV infected cell line.

A study by electron microscopy of HUT 78 cells infected with the ARV-2 strain of HIV-1 revealed, in addition to virions having the characteristic morphology of a lentivirus, the presence of numerous C-type particles, suggesting that the analysed specimen might in fact contain two different viruses. In order to further investigate this observation, the cell culture supernatant was filtered and mixed at serial dilutions with uninfected HUT 78 cells. In this way, it was possible to obtain cells producing only virions with C-type morphology and a Mn++ dependent reverse transcriptase activity which in a sucrose gradient was found to peak at a buoyant density of 1.16 g/cm3. The RNA purified from the culture medium was not detected by reference DNA probes for either HIV-1 and -2 or HTLV-I and -II. In an indirect immunofluorescence assay, sera from patients seropositive for these human retroviruses failed to recognize any antigen in the cells producing only the C-type particles. Using the same technique, plasma samples from 100 blood donors also gave negative results. The presence of this retrovirus could be the result of previous laboratory contamination but the possibility that it is indeed a human virus has to be considered.

Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon.

We present the nucleotide sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions. This DNA sequence derives from the lambda gal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements. We define the precise 3' junction between the bacterial and phage sequences in lambda gal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage lambda. Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator. Lastly, we compare the galK genes of E. coli and the yeast S. cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.

Expression of galactokinase as a fusion protein in Escherichia coli and Saccharomyces cerevisiae.

Plasmids are described that allow fusions between the Escherichia coli galK gene (coding for galactokinase) and any gene of interest. An example is given in which a galK gene, lacking the normal initiator methionine codon, is fused to various segments of the 5' end of the tetR gene of pBR322. The resulting plasmids complemented an E. coli galK mutant, and galactokinase activity was retained despite the addition of up to 250 foreign amino acids to the amino-terminus of the galactokinase polypeptide. In a second experiment, the galK gene was fused to the LEU2 gene of Saccharomyces cerevisiae. The resulting plasmid was able to complement a yeast GAL1-mutant and galactokinase synthesis in yeast was controlled, via the LEU2 regulatory system, by the levels of leucine and threonine in the growth medium. The galK fusion plasmids should facilitate analysis of the control systems of a wide variety of genes in different organisms.