Telomerase targeting by retinoids in cells from patients with myeloid leukemias of various subtypes, not only APL.

Numerous strategies have been proposed to specifically inhibit telomerase (human telomerase reverse transcriptase (hTERT)) but to date only a few are clinically relevant in anticancer therapy. Recently, we have shown that long-term treatment with all-trans retinoic acid (ATRA), a compound clinically approved for differentiation therapy of acute promyelocytic leukemia (APL), represses hTERT in differentiation-resistant APL cell lines leading to telomere shortening and death. This signaling requires the co-activation of the retinoic acid receptor alpha (RARalpha) and the retinoic X receptor (RXR). In contrast to differentiation-therapy, which is only successful in this subtype of leukemia, the telomerase-targeted pathway could also be of use in non-APL. Here, we demonstrate that repression of hTERT occurs in fresh blasts cells from patients with myeloid leukemias of various subtypes exposed ex vivo to ATRA or synthetic retinoids. These results support the idea that, by hTERT targeting, retinoids can induce telomere shortening and cell death and their integration in therapy protocols for myeloid leukemias refractory to maturation should be considered.

Estramustine resistance correlates with tau over-expression in human prostatic carcinoma cells.

Estramustine (EM) is an anti-microtubule drug used in the treatment of hormone-refractory advanced prostate cancer. Since microtubules are the targets for EM cytotoxicity, we investigated the effects of EM on the microtubule-associated protein tau to determine what role it may play in drug resistance. We have compared tau expression in human prostate cancer cells (DU145) and an EM-resistant derived cell line (E4). Reverse transcriptase polymerase chain reaction has established that tau is expressed in both cell lines but increased 1.9-fold in E4 compared with DU145 cells. This result was confirmed at the protein level by Western blotting. Tau is a phosphoprotein, most of its reported phosphorylation sites being serine or threonine residues. We have shown, however, that tau is also phosphorylated at tyrosine residues in DU145 cells and that the phosphotyrosine level of tau is significantly increased in E4 cells. Moreover, DU145 cells exposed to short term micromolar drug concentrations enter a phase of microtubule depolymerization, display an increased level of tau phosphorylation and follow a pattern similar to that observed in EM-resistant E4 cells. EM is therefore able to induce a very rapid change in the posttranslational state of tau. Our results show that the acquisition of EM resistance in E4 cells, which is accompanied by changes at the tubulin level, is also associated with important changes in tau expression and phosphorylation.

Characterization of in vitro culture of HIV-negative Kaposi's sarcoma-derived cells. In vitro responses to alfa interferon.

We established long-term cultures from skin tumors of nine patients suffering from classical Kaposi's sarcoma (KS). Spindle cells obtained after enzymatic digestion were cultured on gelatin- or fibronectin-coated flasks in DMEM with 15% fetal calf serum, aFGF and heparin. Immunohistochemical staining was positive for MHC class I, laminin, type IV collagen, vimentin, alpha smooth muscle actin (20-40% of cells), caldesmon (20%), calponin (20-40%) and smooth muscle myosin (20-40%), and was negative for common leukocyte antigen, CD4, LFA1, CD34 and cytokeratin. Around 20% of cells up to the third passage in culture expressed the endothelial markers CD36, BMA 120 but were negative for UEA and Fc von Willebrand. Smooth muscle proteins were detected with immunoblotting. Using the polymerase chain reaction, human herpes virus 8 (HHV8) sequences were detected in primary cultures of three out of seven cell lines but were rapidly lost during in vitro passaging. KS-derived cells did not proliferate in serum-free medium, had a normal karyotype and did not grow in soft agar medium. Tumors formed in nude mice injected with KS-derived cells. The tumors were composed of mouse cells and were highly vascularized. Our results suggest that KS-derived cells are heterogeneous: the majority of cells have either a smooth muscle cell or a fibroblastic phenotype. Another minor cell compartment was composed of endothelium-derived cells. KS cells do not possess the characteristics of transformed cells in vitro and may be composed of polyclonal activated cells. Recombinant alpha interferon (rIFN) slightly inhibited the growth of KS-derived cells and increased the expression of MHC class I antigens. While cells were resistant to natural killer (NK) cell-mediated cytotoxicity, they became sensitive to rIFN-primed NK cells. Thus, the antitumor potential of rIFN against KS in vivo could result from immunomodulatory rather than from direct antiproliferative effects.

Value of the preoperative detection of prostate-specific-antigen-positive circulating cells by nested RT-PCR in patients submitted to radical prostatectomy.

OBJECTIVE: To assess the value of the detection of circulating prostate cells [prostate-specific antigen (PSA) positive] by reverse-transcriptase nested polymerase chain reaction (nested RT-PCR) to improve the staging of clinically localized prostate cancer. METHODS: Nested PCR was performed on blood samples of 29 patients submitted to radical prostatectomy for clinically localized (T1-T2) prostate cancer. Nine patients with various benign urologic diseases comprised the negative control group. Incubation was for 25 cycles for each PCR, using beta 2-microglobulin to test the integrity of RNA samples. Each sample was tested in quadruplicate and analyzed by agarose gel electrophoresis, blotted and hybridized with specific internal primers. Nested PCR results were compared with the pT stage of the prostate specimen, processed according to the Stanford method. RESULTS: In 6 out of 29 patients (20.7%) with clinically localized prostate cancer, circulating prostate cells were detected by nested PCR. There was no relationship between pathologic stage and RT-PCR results. Eleven out of 14 pT2 patients (78.6%) were PCR negative and only 3 out of 15 pT3 patients (20%) were PCR positive. All control samples were PCR negative. CONCLUSIONS: In selected patients with T1-T2 prostate cancer, there was no relationship between pathologic stage and the presence of circulating PSA-positive cells detected by nested PCR. However, in 20.7% of patients with clinically localized prostate cancer, circulating prostate cancer cells were detected. A further follow-up based on PSA is necessary to clarify the clinical relevance of this biologic anomaly.

Stromal cells from human benign prostate hyperplasia produce a growth-inhibitory factor for LNCaP prostate cancer cells, identified as interleukin-6.

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal-cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial-cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU145 and an SV40 large T-immortalized normal epithelial-cell line, PNT1A, in double-diffusion co-culture chambers. PNT1A was stimulated by PNX (x1.6) and more strongly by BH101 stromal cells (x2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101-conditioned medium (CM), obtained in serum-free conditions, induced 90% inhibition of [3H]thymidine incorporation of the LNCaP androgen-sensitive cell line. Two other androgen-independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL-1beta, IL-6 and IL-8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL-6. IL-6 was demonstrated to be responsible for the strong inhibitory effect since an IL-6-neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL-6. Recombinant IL-6 growth inhibition was observed only on LNCaP prostate cancer androgen-sensitive cells.

Decreased IL-1 beta and TNF alpha secretion in long-term bone marrow culture supernatant from Fanconi's anaemia patients.

Recent studies have shown that abnormalities of cytokine and lymphokine secretion are involved in the pathophysiology of Fanconi's anaemia (FA). In the present study, we quantified IL-1 beta, IL-1 receptor antagonist (IL-1Ra), IL-6 and TNF alpha protein levels in the supernatant of long-term cultures generated from BM cells of FA patients. Cell-free conditioned medium from long-term bone marrow culture was harvested every week at confluence and tested for interleukin secretion. IL-1 beta, IL-1Ra, TNF alpha and IL-6 protein secretion was assessed using immunoassays. IL-6 secretion was similar between controls and FA supernatants from wk 1 to wk 4. TNF alpha released from FA cells was consistently found at very low levels compared to control cells during the first 3 wk. Furthermore, secretion of IL-1 beta by cells from FA was always more than 2 standard deviations below the value of IL-1 beta found in normal donor cells from wk 1 to wk 4. In conclusion, in addition to a stem cell defect, a marked decrease in IL-1 beta and TNF alpha secretion may be one of the mechanisms leading to bone marrow failure in individuals with Fanconi's anaemia.

Radioimmunoassay of erythropoietin: analytical performance and clinical use in hematology.

We report here the performance of a recently commercialized radioimmunoassay kit for determining erythropoietin (EPO) in serum or plasma. The lower detection limit of the method was 3 U/L. Precision, analyzed by the variation coefficients between different assay runs and in the same experiment, was always less than 10%; accuracy was assessed by recovery and dilution tests. In anemic patients (hematocrit 18-39%), the concentration of EPO was logarithmically related to hematocrit. A relatively large dispersion of the results was noted, as reported by others with various RIAs. Patients with severe renal failure demonstrated a very low EPO value, whatever the degree of their anemia. In some chronic anemias resulting from malignancy, EPO concentrations were also relatively low. In the polycythemia vera group, the EPO mean was below normal for greater than 95% of the patients, whatever their clinical stage (first evaluation, relapse, or remission). In contrast, 91% of the patients with pure erythrocytosis had a normal or increased EPO value, even when the etiology was unknown. Measurement of EPO concentration may be useful for the clinical differentiation of myeloproliferative disorders and, subsequently, for their prognosis and choice of treatment.

Radioimmunoassay of immunoreactive erythropoietin as a clinical tool for the classification of polycythaemias.

Radioimmunoassay of erythropoietin (EPO) has been used for evaluating its clinical usefulness in distinguishing polycythaemia vera (PV) from pure erythrocytosis (PE). A normal log distribution (13.40 mU/ml +/- 2.45) was observed in the 66 reference samples. Similar results were observed in 29 pure thrombocytaemias (13.23 +/- 5.19). In PV patients, whether in clinical remission or in active phase, the EPO titer was lower than normal values (respectively 7.32 +/- 3.63 and 6.59 +/- 2.75), without any correlation with the haematocrit (44 to 51% and 52 to 71%). In the anaemic cases (excessive therapy, spent phase, myelofibrosis), a slight excess of EPO titer was observed, but less than expected when taking the hematocrit into account. In contrast, pure erythrocytosis is a very heterogeneous group, with the highest EPO values in the well-defined secondary cases, and normal or slightly excessive values in most of the cases, but with some low values similar to those observed in PV cases. In term of PV diagnosis, a 90% predictive value is observed when a "cut-off" value of 11 mU/ml is chosen. When the "cut-off" is 16 mU/ml, no PV case was observed beyond this value. We conclude that EPO assay is useful in myeloproliferative diseases from a practical point of view.