Prognostic value of cytokeratin 19 fragment (CYFRA 21-1) and cytokeratin-positive cells in bone marrow samples of breast cancer patients.

The aim of this study was to investigate the relationship between the detection of micrometastatic cells by immunocytochemistry (ICC) with an anticytokeratin antibody and cytokeratin fragment (CYFRA 21-1) expression detected by an immunofluorescent assay in bone marrow of breast cancer patients. Micrometastatic CK+ cells were screened with a pancytokeratin antibody A45 B/B3 from bone marrow aspiration samples of 102 breast cancer patients (65 primary tumors, 10 local recurrences and 27 distant metastases). CYFRA 21-1 levels were assessed in bone marrow supernatant of these patients before collection of the mononucleated interface cells on a Ficoll-Hypaque density gradient and in 20 control patients. CYFRA 21-1 and CK+ cell detection by ICC were both correlated with clinical stage. CYFRA 21-1 was significantly elevated in patients with micrometastatic disease detected by ICC: 4.77 ng/mL (+/- 10.87 SD) versus 1.00 ng/mL (+/-1.36 SD) in patients with negative ICC (p=0.01). In univariate analysis, a CYFRA 21-1 value > or =1 ng/mL and the presence of CK+ cells were associated with a poorer survival for patients with stage I to III breast cancer (n=65). On multivariate analysis, only pathological nodal status and presence of CK+ cells in bone marrow were independent prognostic factors for overall survival. In conclusion, in this series CYFRA 21-1 was correlated with detection of CK+ cells by ICC in bone marrow, but cannot replace ICC. The presence of CK+ cells in bone marrow remains a strong independent prognostic factor in primary breast cancer.

Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer.

There is increasing statistical evidence that the presence of tumour cells in bone marrow detected by immunocytochemistry represents an important prognostic indicator in breast cancer, but their individual capacity to become clinical metastases is unknown. The aim of this study was to assess the proliferative capacity of these occult metastatic cells in the bone marrow of patients with various stages of breast cancer. We obtained bone marrow aspirates from 60 patients with breast cancer before treatment with chemotherapy: 17 stage II, 12 stage III and 31 stage IV. After bone marrow culture for 6-34 days (median: 17 days) under specific cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 40 patients (66%). Expansion of tumour cells was poorly correlated with tumour cell detection on primary screening (P=0.06). There was a nonsignificant correlation between the number and the presence of expanded tumour cells and the UICC stage of the patients. On primary screening, tumour cell detection was positive in 56% of patients and was correlated with clinical UICC stage (P=0.01). However, with a median follow-up of 23 months, expansion of tumour cells from bone marrow was associated with decreased patient survival (P=0.04), whereas the survival difference according to detection of CK-positive cells on primary screening was not statistically significant. In conclusion, viable tumour cells can be detected in the bone marrow of breast cancer patients. Their proliferative potential could be predictive of outcome and deserves further investigation.

Illegitimate villin transcripts in normal bone marrow precludes detection of colon cancer micrometastases.

Villin is a specific marker for normal and tumoral colon tissue. We have developed a highly sensitive assay using reverse transcription (RT) and real-time PCR to detect villin transcripts. The sensitivity of detection is one colon cancer cell. However, high levels of illegitimate villin transcripts were observed in normal bone marrow, precluding the use of villin RT-PCR for routine detection of colon cancer cells in bone marrow of patients with colon cancer.

Relationship between cell migration and cell cycle during the initiation of epithelial to fibroblastoid transition.

The NBT-II rat bladder carcinoma cell line, which displays epithelial to mesenchymal transition or EMT in response to FGF-1 stimulation, was used to study the interrelationships between cell cycle and cell scattering and locomotion. Time-lapse video microscopy experiments were performed with asynchronous growing cells and lovastatin-arrested cells. FGF-1 stimulation induced cell movement in cells in all phases of the cell cycle, except G2 + M phase, in which cells did not respond to stimulation. The delay between cell stimulation and cell movement depended on the age of the cell at the beginning of cell stimulation: cells less than 4 h old when stimulated by FGF-1 had a 1-h delay whereas cells more than 4 h old had a 3-h delay. Cells stimulated before they were 4 h old were temporarily arrested in their cell cycle progression. Older cells underwent mitosis on schedule. Lovastatin-treated cells were shown to be synchronized in the G1 phase and to migrate simultaneously after FGF-1 stimulation. These results indicate that the G1 phase was a critical phase for FGF-1 induced cell migration during epithelial to fibroblastoid transition.

[Current data on the role of APC protein in the origin of colorectal cancer]. / Données récentes sur le rôle de la protéine APC dans la genèse du cancer colorectal.

The adenomatous polyposis coli (APC) gene has been found to be mutated during the development of sporadic colorectal cancers as well as in familial adenomatous polyposis (FAP). These conditions result from initially somatic and germ line mutations respectively. In both cases, the expressed protein is truncated at its carboxyterminal region. Investigations into the role of wild-type APC have led to a better understanding of the importance of mutations in the genesis and progression of adenomas. APC was shown to regulate cell growth and cell death, to bind beta-catenin, and to colocalize with microtubules. APC truncation was therefore hypothesized to alter cell multiplication and cells are no longer able to undergo apoptosis. Owing to its beta-catenin binding, APC can modify the pool of beta-catenin which is in part utilized in the assembly of adherens junctions and in nuclear signalling. Truncated APC is unable to regulate this pool thereby altering adhesion and cell signalling. Finally, APC involvement in microtubule-dependent locomotion may explain some changes in cell movement which are observed in adenomas. The establishment of murine mutants and of normal and malignant intestinal cell cultures have allowed to assess biochemical and physiological properties of APC and its putative role in the genesis of colorectal carcinogenesis. Moreover, these experimental models have suggested a variety of possible therapeutic approaches.

The APC gene product and colorectal carcinogenesis.

The adenomatous polyposis coli (APC) gene has been found to be mutated during the development of sporadic colorectal tumours as well as in familial adenomatous polyposis patients (FAP), mutations being somatic or germinal respectively. The gene product is truncated in the carboxyterminal region but the role of the APC protein in tumorigenesis is not well understood. The purpose of this review is to reassess studies on the APC protein in an attempt to understand how the loss of its functions may cause or contribute to the development of carcinomas.

Nuclear retinoic acid receptor characterization in cultured human trophoblast cells: effect of retinoic acid on epidermal growth factor receptor expression.

Vitamin A is an important factor during gestation and its metabolite, retinoic acid (RA), is a potent teratogen. However, RA action on the placenta is still poorly understood. In this study we analysed the presence of RARs and RXRs in human trophoblastic cells. We determined that RAR alpha was the more expressed form in term placenta, and that RAR beta was induced by RA treatment. Then we analysed RA effects on endocrine activities and on epidermal growth factor (EGF) receptor expression. We found that RA decreased 125I-labeled EGF binding and EGF-dependent phosphorylation. Furthermore, RA treatment led to a concentration-dependent decrease in the amount of EGFR protein expression. This treatment also decreased EGF receptor mRNA levels, suggesting transcriptional regulation of the EGF receptor. Thus we demonstrated that RA could interact with feto-placental development by modulating trophoblast EGF receptors expression, probably via its nuclear receptors.

Uterine cells other than stromal decidual cells are required for 1,25-dihydroxyvitamin D3 production during early human pregnancy.

Human decidual cells are known to produce 1,25-(OH)2D3 at the end of pregnancy, the present study evaluates this capacity, and the part played by stromal decidual cells, in early pregnancy. Cells were obtained from nine human decidua by aspiration or curettage during early pregnancy (7-10 weeks), separated on Ficoll-Paque and plastic adherence, and incubated for 1 h with 25-(OH)D3. Incubation medium and cells were extracted and chromatographed on two successive HPLC systems. The cells examined were of both physiological and pathological (ectopic pregnancy) origin. Endometrial cells obtained in four non-pregnant situations (myomas) were also studied to determine whether the 1,25-(OH)2D3 synthesis by the uterus is associated with the appearance of decidual cells. Results show that human decidual cells from early pregnancy convert 25(OH)D3 (2.5 nM or 2.5 microM) into a metabolite with the physicochemical characteristics of synthetic 1,25-(OH)2D3. This ability is shared by cells isolated during early pregnancy, whether physiological or ectopic (tubal pregnancy). Non-adherent cells, which include mainly stromal decidual cells, are less able to produce 1,25-(OH)2D3 than are the adherent cells, suggesting that macrophages, granulocytes or as yet unidentified cell types are required for the 1,25-(OH)2D3 production by decidual tissue during early human pregnancy. In addition, one out of four experiments with non-pregnant endometrial cells could produce 1,25-(OH)2D3 suggesting that, although not the rule in the non-pregnant state, in vitro production of 1,25-(OH)2D3 by uterine cells can be found in the absence of decidual cells.

Modulation of pp60c-src activity and cellular localization during differentiation of human trophoblast cells in culture.

The morphological and functional differentiation of human trophoblast cells ends with the formation of terminally differentiated multinucleated syncytial trophoblasts. This in vivo differentiation is mimicked in vitro during the primary culture of extravillous cytotrophoblasts: isolated mononuclear cytotrophoblasts aggregate and fuse to form syncytia. This in vitro differentiation is associated with an increase in epidermal growth factor receptor (EGF-R) expression and a transitory increase in E-cadherin expression during cell aggregation. In the present study, we investigated the expression of pp60c-src during morphological differentiation of trophoblast cells. Cultures were terminated at various time intervals and pp60c-src was analysed by immunocytochemistry using a specific antibody. In addition, pp60c-src was investigated by western blot analysis and its tyrosine kinase activity was measured concomitantly. In mononuclear cytotrophoblasts, pp60c-src was localized at cell-matrix contacts and during the aggregation of cytotrophoblasts, pp60c-src was distributed on the cell surface at points of cell-cell contact being colocalized with EGF-R and E-cadherin. The kinase activity of the pp60c-src protein increased significantly at day 2 when cells were completely aggregated and started to fuse, and remained elevated while cells underwent further differentiation. Inhibition of pp60c-src by herbimycin A at 0.25 to 1 microgram/ml during the first day of culture was associated with a decreased expression of tyrosine kinase activity of EGF-R and an increase in E-cadherin expression. These data suggest that pp60c-src is involved in the modulation of trophoblast cell aggregation and fusion leading to syncytial formation.

Effects of calcitonin on human trophoblastic cells in culture: absence of autocrine control.

The purpose of this work was to investigate the effects of calcitonin (CT) on trophoblastic cells with respect to cAMP levels and human chorionic gonadotrophin (hCG) secretion in cultured cells from first-trimester and term placentas and in a choriocarcinoma cell line (JEG-3). The expression of the CT gene was investigated to elucidate a putative autocrine control of CT during pregnancy. The addition of salmon CT (10(-10) M and above) resulted in concentration-dependent increases in cAMP secretion by normal trophoblastic cells from term and first-trimester placentas. Moreover, CT was found to increase cAMP secretion preferentially in completely differentiated cells, i.e. after 4-7 days in culture. Addition to the culture medium of JEG-3 cells slightly increased cAMP secretion only at a concentration of 10(-8) M. The basal level of hCG in the medium was found to be higher in the first-trimester than in the term trophoblast culture, but salmon CT induced an increase in hCG secretion by term placenta cells only. CT gene expression in our experimental model was investigated to elucidate a putative autocrine control of CT action during pregnancy. It was not found to be expressed in syncytiotrophoblast cells from either first-trimester or term placenta cells by the method used. Our data demonstrate the absence of autocrine control of CT effects in trophoblastic cells, and suggest that CT is likely to exert its effect preferentially on differentiated cells.

Effect of calcitriol on peripheral blood lymphocyte cytotoxicity.

To clarify the biochemical mechanism responsible for the inhibition by calcitriol (1,25-dihydroxyvitamin D3) of cytotoxicity in peripheral blood lymphocytes (PBL), the human NK sensitive K562 cell line and the human tumor necrosis factor-sensitive murine L929 cell line were used as targets and subsequently compared. The cytotoxicity of PBLs for K562 cells was not changed by preincubation for 4 h with 10 ng/ml phorbol myristate acetate (PMA), but was reduced after an overnight preincubation with 10(-9) or 10(-8) M calcitriol. Using L929 cells, preincubation of PBLs with 10 ng/ml PMA for 4 h increased their cytotoxicity. Overnight incubation with calcitriol significantly reduced PBL cytotoxicity for L929 cells in a dose related manner and suppressed the enhancement of this cytotoxicity by PMA. Pretreatment of PBLs with cycloheximide reduced their cytolysis for L929 cells but did not change their cytotoxicity towards K562 cells. Consequently, the natural cytotoxicity of PBLs for K562 cells does not involve the same mechanism as their cytotoxicity for L929 cells and is therefore subject to different forms of regulation. However, calcitriol reduced PBL cytotoxicity towards both target cells.

Effects of 1,25-dihydroxyvitamin D3 on in vitro lymphocyte reactions: arguments for a role at the maternofetal interface.

Calcitriol or 1,25-dihydroxyvitamin D3 is synthesized by successive hydroxylations of vitamin D in the liver and kidney and during pregnancy in the placenta and the decidua. The aim of the present study was to clarify the immunoregulatory role of calcitriol, if any, during pregnancy. Calcitriol concentrations of 10(-10) to 10(-8) M was shown to reduce the proliferation of allogeneically stimulated lymphocytes and cytotoxic cell generation in a dose-dependent manner. However, calcitriol did not inhibit IL-2-dependent proliferation of CTLL-2 cell line. Calcitriol reduced non-MHC restricted cytotoxicity. Calcitriol, therefore, might be involved in the successful engraftment and growth of the fetoplacental unit possibly synergized with other products of placental or decidual origin.

Phorbol myristate acetate enhances adenylate cyclase activity in human polymorphonuclear leukocytes.

The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol.

Superoxide anion production in glass-adherent polymorphonuclear leukocytes and its relationship to calcium movement.

The production of superoxide anion (O2-.) was measured in relation to 45Ca movement in glass-adherent polymorphonuclear leukocytes (PMNs), and the results were compared with those obtained by ourselves and others on PMNs in suspension. In adherent PMNs, O2-. production stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was of rapid onset and short duration; this was also true of PMNs in suspension. However, O2-. production was insensitive to the concentration of extracellular calcium. Both in adherent and non-adherent PMNs, O2-. production stimulated with phorbol myristate acetate (PMA) had a latency time and was of long duration. In adherent PMNs, pretreatment with PMA potentiated the FMLP-induced O2-. production by lengthening its duration without changing its initial rate. In adherent PMNs (10(-10)-10(-7) M) FMLP induced a fast but transient dose-dependent increase in 45Ca within 1 min, whereas PMA only released 45Ca about 5 min after its addition to the cell culture medium. Pretreatment of PMNs with 10 or 100 ng/ml PMA for 3 min before stimulation by 10(-7) M FMLP reduced the 45Ca efflux observed with FMLP alone. We conclude that O2-. production by adherent PMNs cannot simply be related to Ca2+ movement. In comparison with non-adherent cells, adherence seemed to interfere with the characteristics of both calcium and O2-. generation, probably by modifying the cytoskeleton.

Effect of maternal insulin deficiency on vitamin D metabolite concentrations in normoglycemic pregnant rats and their fetuses.

The effect on vitamin D metabolite concentrations of insulin deficiency, not accompanied by hyperglycemia, were investigated in pregnant rats and in their fetuses injected with 75 mg/kg BW streptozotocin (SZ). These concentrations were measured in maternal plasma and whole fetal body. In the insulinopenic mothers, the 25OHD concentration was found to rise compared with that of control pregnant rats (7.00 +/- 1.66 ng/ml, n = 16, versus control 4.50 +/- 1.60, n = 10, 0.001 less than P less than 0.01). The concentration of 1,25(OH)2D, which was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was previously found to decrease in pregnant rats that were both hypoinsulinic and hyperglycemic, was not different in our control and insulinopenic rats (107.36 +/- 38.25 pg/ml, n = 11, versus control 122.90 +/- 18.20, n = 18.20, n = 8). In fetuses from our SZ-injected rats, the 24,25(OH)2D level diminished compared with the control level (2.12 +/- 0.70 ng/g, n = 11, versus control 5.23 +/- 0.95 ng/g, n = 13, P less than 0.001). The Ca/P ratio in fetal body also decreased (0.68 versus control 1.12). It is suggested that the placental metabolism is an important determinant or normal fetal growth.

Calcium-transport in quiescent and 1,25-dihydroxycholecalciferol-stimulated human osteosarcoma cells. Role in 24 hydroxylase enhancement.

The present study evaluates in osteosarcoma cells, the effects of a calcium channel inhibitor nicardipine in 24-hydroxylase activity and 45Ca desaturation curve in presence of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). This sterol induced an increase in 24-OHase activity and 45Ca fluxes. Nicardipine reversed the effect of 1,25(OH)2D3 on 45Ca fluxes but reinforced the enhancement of the 24-OHase activity. The fact that the effects of 1,25(OH)2D3 were reduced by cycloheximide support the hypothesis of a de novo protein synthesis. Our study has allowed us to dissociate the effects of 1,25(OH)2D3 on 24-OHase enhancement from those on Ca2+ transport.

Effects of experimental diabetes on the vitamin D metabolism of pregnant rats and their fetuses.

The effects of streptozotocin-induced diabetes on the vitamin D metabolism of pregnant rats were investigated in mothers and their fetuses, 11 and 14 days after streptozotocin (SZ) injection, i.e., on days 18 and 21 of gestation. In the mothers' plasma, the levels of 25-hydroxycholecalciferol (25OHD) and 1,25-dihydroxycholecalciferol (1,25(OH)2 D) were not different from control levels on day 18, but on day 21, 25OHD had increased, 1,25 (OH)2 D had diminished, and significant hypercalcemia was noted (10.1 +/- 0.27 mg/dl vs. 9.47 +/- 0.19 mg/dl, mean +/- SD). In hyperglycemic fetuses from the diabetic mothers, plasma insulin levels were reduced at day 18 but enhanced at day 21. 25OHD levels were not different from those of the controls at day 18, but were lower at day 21 (2.12 +/- 0.70 ng/g BW, n = 13, vs. 3.75 +/- 1.40 ng/g BW n = 29 controls, means +/- SD). Fetal body levels of 1,25 (OH)2 D were lower than that in the controls at day 18 (16.6 +/- 2.9 pg/g BW, n = 9 x 2, vs. 28.7 +/- 6.3 pg/g BW, n = 7 x 2, mean +/- SD P less than 0.001), but identical to control levels on day 21. The role of fetal or placental enzymes in the regulation of vitamin D metabolism in fetuses is discussed.

Comparative effects of the ionophore A23187 and vitamin D metabolites on 45Ca desaturation curves derived from bone cells: interaction of a calcium channel channel inhibitor diltiazem.

The effects of diltiazem, a calcium channel inhibitor, on the cellular transport of calcium were studied in isolated heterogenous rat bone cells. Efflux was measured after equilibrating the cells with 45Ca and adding the vitamin D metabolite (1,25dihydroxycholecalciferol-1,25(OH)2D3 or 24,25dihydrocholecalciferol-24,25(OH)2D3), the ionophore A23187 and/or diltiazem. Results were analysed by fitting the desaturation curve to a model of two exponential terms. Kinetic analyses of curve indicated the presence of 2 exchangeable pools with different rate constants of exchange between the medium and cells (expressed by K.). After incubation of bone cells with diltiazem (20 nmol/10(6) cells) the following changes were recorded: a marked decrease in the rate constant of efflux from the fast turnover calcium pool (K12) and a reduction of the calcium pool sizes. Incubation of 10(6) cells with 0.5 ng 1,25(OH)2D3 plus diltiazem significantly reduced K12 compared to incubation with 1,25(OH)2D3 alone. In presence of 24,25(OH)2D3, diltiazem did not significantly alter K12 which was raised by incubation with the metabolite alone. Ionophore A23187 (0.5 micrograms/10(6) cells) increased the value of slow turnover constants of efflux whose values were affected by diltiazem. The possible involvement of Ca movements in bone resorption does not seem confirmed in the present experiment since in vitro effects of diltiazem in organ culture (observed in an initial previous experiment) were not reflected in the calcium 45 desaturation kinetics in heterogenous bone cells.

[Role of the placenta in maintaining the fetal allograft]. / Rôle du placenta dans le maintien de l'allogreffe fétale.

Present knowledge of the alloantigenic status of the placenta, which makes it a natural semi-allogeneic allograft is briefly surveyed. The systemic immunoregulatory mechanisms operating during normal allopregnancy which are not a prerequisite for a successful pregnancy are recalled. The placenta--dependent local mechanisms, e.g. trophoblast dependent decidual suppressor cells, factor mediated and factor independent resistance to cell mediated lysis, are surveyed as well as some of the mechanisms of action of trophoblast regulatory factors, namely suppressor cell induction and inhibition of IL-2 dependent cell growth/activation. The main feature of the CBA x DBA/2 model of spontaneous abortions in mice, and its prevention by anti Balb/c leukocyte immunisation are described. It was shown that anti-T cell depletion prevents the anti-abortive effects of immunisation. Such a treatment is also able to restore normal placental weight in auto-immune MRL lpr mice, which are known to display excess seric CSF beta-like activity (CSFs being in vitro efficient growth factors for trophoblasts). Transfer of such cells into the MHC compatible CBA/J prevents resorbtions upon a subsequent mating with DBA/2. Thus, direct effects of CSFs beta 1 and beta 2 as anti abortifacient (IL3 and GM CSF) are described together with the abortificacient effects of TNFs and NKs activators.