Simian virus SV40 has been widely used to immortalize epithelial cells of mammalian origin. We report here, for the first time to our knowledge, the immortalization of normal adult prostatic epithelial cells in culture by transfection of a plasmid containing SV40 genome with a defective replication origin (SV40 ori-) encapsulated into liposomes. These cells (PNT1) have now been cultured for more than 12 months, and shown to contain the SV40 genome. They express large T protein, present the phenotype of differentiated luminal prostatic cells (positive with antibodies to cytokeratin 18, 19, weakly positive for prostatic acid phosphatase and prostatic specific antigen, negative with anticytokeratin 14 and KL2 antibody). PNT1 cells contain high affinity receptors for dihydrotestosterone. These cells provide a useful tool to study the biology and the pathology of adult prostatic epithelial cells, specially to understand the steps leading to prostatic transformation.

SIAH-1, the human homologue of the drosophila seven in absentia gene, is a p53-p21 Waf-1 inducible gene. We report that stable transfection with SIAH-1 of the epithelial breast cancer cell line MCF-7 blocks its growth process. The transfectants show a redistribution of SIAH-1 protein within the nucleus, more speci®cally to the nuclear matrix, associated to dramatic changes in cell morphology and defective mitosis. Multinucleated giant cells (2 ± 12 nuclei in more than 50% cells) were a most striking observation associated with tubulin spindle disorganization and defective cytokinesis. There were also present at high frequency abortive mitotic ®gures, DNA bridges and persistance of intercellular bridges and midbodies, along with an increased expression of p21 Waf-1 . These results indicate that the mechanism of growth arrest induced by SIAH-1 in MCF-7 cells involves disorganization of the mitotic program, mainly during nuclei separation and cytokinesis.

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21 WAF-1 , by inf luencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21 WAF-1 resulted in expression of the HUMSIAH (human seven in absentia homologue), Rb, and Rbr-2 genes and strong suppression of the malignant phenotype. p21 WAF-1 drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and f luorescence in situ hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.

Summary Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (X2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (X2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.

The spontaneous expression of HLA class-I and class-II molecules in 5 human breast carcinoma cell lines, MCF-7, T47D, ZR75-1, HSL-53, MDA-MB 231, and their modulation during IFN-gamma treatment, are reported. The expression of cell-surface determinants was examined by indirect immunofluorescence using monoclonal antibodies (MAbs) specific for HLA class-I and class-II (DR, DQ and DP) antigens. The biosynthesis and maturation of these molecules were analyzed by 2-dimensional gel electrophoresis analysis (2D-PAGE) of class I, DR alpha, beta and invariant immunoprecipitates. Transcription was analyzed by Northern blot hybridization with HLA class-I and -II cDNA-specific probes. In all cell lines, more than 80% of cells expressed HLA class-I antigens at their surface. 2D-PAGE and mRNA studies showed a variable basal level of HLA class-I biosynthesis and transcription with a constant increase after 1,000 U/ml IFN-gamma treatment. HLA class-II determinants were totally absent from the surface of MCF-7, MDA MB231, ZR75-1 and T47D but they were detected in a small subpopulation of HSL-53 cells (DR 6%, DQ 6%, DP 20%). Spontaneous biosynthesis of HLA-DR molecules in immunoprecipitates analyzed by 2D-PAGE or transcripts in Northern blot were not detected in the 5 cell lines. Treatment with 1000 U/ml IFN-gamma induced or increased the expression of HLA class-II molecules in all cell lines but DQ expression was variable. While T47D, ZR75-1 and HSL-53 increased their transcripts and antigen expression, MDA, MB231 and MCF-7 showed no DQ mRNA transcript. Biochemical analysis of the DR products revealed a classical alpha, beta and invariant (li) chain pattern, but indicated a constant glycosylation defect in the invariant chain in all cell lines, associated with weak expression of the beta chain and the presence of an extra spot of low molecular weight in the acidic part of the gel. Thus, post-transcriptional events did not appear to be totally controlled by IFN-gamma in the different cell lines. These differences in DQ expression and glycosylation process in different breast cancer cells may be important in the activation of the immune response among different individuals.

Granylocyte colony forming units (CFU-C) were studied in 22 patients with severe aplastic anaemia before and after treatment with antilymphocyte globulin (ALG). Nine patients showed a clinical response to ALG characterized by a rise in the granulocyte count to over I X 10(9)/1 within 30 d. These patients were distinguished in vitro from non-responders by an increase in CFU-C numbers after incubation of bone marrow cells with ALG, and by the presence of inhibitors of normal CFU-C in the serum in six out of seven patients tested. In responding patients bone marrow CFU-C rose while most non-responding patients showed no change or a fall in CFU-C after treatment. In addition in three out of four responding patients examined serum inhibitors disappeared after treatment. The horse ALG used in this study also stimulated normal CFU-C in vitro. This evidence is contrary to the hypothesis that ALG stimulates CFU-C in aplasia by inactivating an abnormal suppressor lymphocyte population. The nature of the stimulation by ALG remains unclear. But in practice the effect of ALG on bone marrow cells and study of CFU-C inhibitors in serum could be used to select patients likely to respond to ALG treatment.

The clinical, hematologic, and immunologic findings of a syndrome of agranulocytosis, hypogammaglobulinemia, and thymoma are described. Neutrophil agranulocytosis predisposing to severe infectious disease resulted from a deficiency of mature cells in bone marrow. Autologous and heterologous stem cell growth in vitro was inhibited by the patient's serum. Immunoglobulin deficiency was secondary to the absence of peripheral blood B lymphocytes, while T-cell subpopulations and cellular immunity were present. Surgical removal of a spindle cell thymoma had no effect on the agranulocytosis and B-cell deficiency. The hematologic findings did not respond to steroid therapy and cyclophosphamide. However, the agranulocytosis improved with repeated plasmapheresis and the patient achieved a clinical remission.