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Biomedical subjects

F Calvo

Publications and source records attributed to F Calvo.

At least 19 recordsLinked to original sources
PubMed1992

The immune response to p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational hot spot.

Alteration of the p53 gene is the most frequent genetic feature of human cancer and leads to overexpression of the altered protein in the tumor cell nucleus. Two diagnostic procedures are currently available to assess p53 mutations: (a) molecular analysis of the gene sequence; and (b) immunohistochemical analysis of p53 protein accumulation. We now report a third approach, serological analysis. Fifteen % of primary breast cancer patients were found to have circulating antibodies to p53 protein by immunoprecipitation or immunoblotting. We have found a close correlation between the presence of such antibodies and bad prognosis such as high histological grade and the absence of hormone receptors. Furthermore, we found that the B-cell response to p53 protein is induced by two immunodominant regions located at the carboxy and amino termini of the protein, outside the central mutational hot spot region. These findings suggest that serological analysis, combined with molecular and histochemical methods, may be suitable for assessing the state of the p53 gene in cancer patients.

Antibodies, Neoplasm↗
PubMed1992

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

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.

Adult↗
PubMed1992

[Role of conservative surgery in the multidisciplinary treatment of Ewing's sarcoma in childhood].

BACKGROUND: In order to cure Ewing's sarcoma it is necessary to have an approach which considers the radical local control on the sites of macroscopic disease, along with the systemic control of micrometastases. On the present study the experience of the authors in analyzed, remarking the role of cytoreduction surgery on curability. METHODS: From January 1982 to August 1991, 24 patients with the mean age 13 years, 14 boys and 10 girls, previously untreated and with a pathology proven diagnosis have been treated by the authors. The treatment protocol included: alternating chemotherapy with cyclophosphamide, adriamycin, methotrexate, bleomycin, actinomycin D and vincristine; administered simultaneously with preoperative external radiation with a volume that completely included the affected bone and surrounding soft tissues for a total dose of 45 Gy/4.5 weeks. After a resting period of 4 weeks, resection of the involved bone and adjacent healthy bone was performed, followed by a single dose of 10-15 Gy of intraoperative radiotherapy to the tumor bed. Subsequently a custom prostheses or allograft was implanted. RESULTS: Twenty patients had localized disease and 4 had metastatic disease at diagnosis. In 16 cases the tumor was in extremities, 5 axial, and 3 extraskeletical. In 15 patients surgery with limb sparing techniques was performed, 8 had en block resection and one amputation (calcaneous location). At the time of this report 21 patient are alive (87%). Four had disease progression, of this 3 had died (12%). The actuarial disease free survival rate is 80% +/- 9% with a follow-up of 104 months, being the mean survival time of 85.5 months. CONCLUSIONS: The cytoreduction surgery included into a multidisciplinary approach permits to achieve a high rate of cure in Ewing's sarcoma. The toxicity of the program can be considered acceptable.

Actuarial Analysis↗
PubMed1992

Tumor necrosis factor-mediated cell lysis in vitro: relationship to cAMP accumulation and guanine nucleotide-binding proteins.

We have investigated the modulation of tumor necrosis factor (TNF)-mediated tumor cell lysis by cAMP. Among a panel of human breast tumor cell lines, MCF7 and MDA MB 231 were shown to be, respectively, sensitive and resistant to TNF-mediated cell lysis in vitro. 125I-labeled TNF-binding experiments demonstrated that both cell lines bind TNF, indicating that the differential sensitivity to TNF was not related to TNF receptor expression. To study the relationship between TNF-mediated cell lysis and cAMP accumulation, cAMP measurement was performed following TNF treatment. Our data show that TNF alone did not induce an enhancement of intracellular cAMP accumulation either in the TNF-sensitive or in the TNF-resistant cell line. Experiments in which cells were exposed to forskolin revealed that this cAMP elevating drug was efficient in enhancing the sensitivity to TNF of MCF7 cell line. This potentiating effect of forskolin was maximal for suboptimal concentrations of TNF (10 ng/ml), reaching up to 100% when forskolin was added at 100 microM. However, co-stimulating with forskolin of either MDA MB 231 or a TNF-resistant MCF7 clone (MCF7-R-A1) did not induce any reversal of resistance to TNF. We further assessed the interaction of TNF with transmembrane signalling and the possible involvement of guanine nucleotide-binding proteins (G-proteins). Bacterial toxin-catalyzed ADP ribosylation of MCF7 and MDA MB 231 membranes was, therefore, performed. Using cholera toxin, we demonstrate that TNF treatment did not quantitatively alter the activity of stimulatory G-proteins either in MCF7 or MDA MB 231 cell line. In contrast, pertussis toxin-catalyzed ADP ribosylation experiments suggest a functional coupling of TNF receptors to a 40-kDa pertussis toxin-sensitive G-protein in the TNF-sensitive MCF78 cell line but not in the TNF-resistant MDA MB 231 cell line. Taken together, these data indicate that cAMP might play a role in TNF-mediated cell lysis and are in support of the involvement of a pertussis toxin-sensitive G-protein in TNF-mediated MCF7 cells lysis.

Adenosine Diphosphate Ribose↗
PubMed1992

Functional expression of VIP receptors in normal, immortalized and transformed mammary epithelial cells.

The effect of VIP and its related peptides on cAMP production has been characterized: 1) in long term culture of normal human mammary epithelial cells (HMEC); 2) in immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40; 3) in the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1D, described to exhibit normal morphogenesis in vivo and functional differentiation in vitro. Basal cAMP levels were increased 1.5- to 8.7-fold in mammary epithelial cells (p less than 0.001-0.05), with a potency EC50 = 0.02-0.6 nM VIP. The pharmacological specificity of the VIP receptors coupled to cAMP generation was established according to the following potency sequence: VIP greater than PACAP-38 greater than helodermin greater than PHM, PHV greater than helospectin 1 much greater than hpGRF, secretin in HMEC, VIP greater than PACAP-38 greater than helodermin greater than helospectin 1, PHM, PHV greater than hpGRF greater than secretin in S1T3 cells, and VIP, PHI, helodermin greater than PHV greater than rhGRF greater than secretin in HC-11 cells. Our data demonstrate the presence of functional, highly sensitive and specific VIP receptors in normal, immortalized and transformed mammary epithelial cells, suggesting a regulatory role for this neuropeptide on the growth, differentiation and function in normal and neoplastic breast tissue.

Animals↗
PubMed1992

Cell and membrane lipid analysis by proton magnetic resonance spectroscopy in five breast cancer cell lines.

The lipid composition of five human breast cancer cell lines (MCF-7, T47D, ZR-75-1, SKBR3 and MDA-MB231) was assessed by proton magnetic resonance spectroscopy (MRS) in whole cells and membrane-enriched fractions. The proportions of the three main lipid resonances in 1D spectra were different for each cell line. These resonances included mobile methyl and methylene functions from fatty acids of triglycerides and phospholipids and N-trimethyl from choline of phospholipids. T47D and ZR-75-1 cells presented a high methylene/methyl ratio (6.02 +/- 0.35 and 6.28 +/- 0.90). This ratio was significantly lower for SKBR3, MCF-7 and MDA-MB231 cells (2.76 +/- 0.22, 2.27 +/- 0.57 and 1.39 +/- 0.39). The N-trimethyl/methyl ratio was high for MDA-MB231 and SKBR3 cells (1.38 +/- 0.54 and 0.86 +/- 0.32), but lower for MCF-7, T47D and ZR-75-1 cells (0.49 +/- 0.11, 0.16 +/- 0.07 and 0.07 +/- 0.03). 2D COSY spectra confirmed these different proportions in mobile lipids. From 1D spectra obtained on membrane preparations, T47D and ZR-75-1 were the only cell lines to retain a signal from mobile methylene functions. These differences might be related to the heterogeneity found for several parameters of these cells (tumorigenicity, growth rate, hormone receptors); an extended number of cases from fresh samples might enable clinical correlations.

Breast Neoplasms↗
PubMed1992

[The role of gastrin releasing peptide as a lung growth factor].

The proliferation of the bronchial epithelium and tumors associated with this tissue is controlled by various growth factors. The main factor is Gastrin Releasing Peptide (GRP), the human counterpart of the amphibian bombesin. These neuropeptides also act as neuromediators and gut hormones. All peptides of this family share a conserved C terminal sequence which is required for biological activity. The determination of this sequence has provided the basis for the design of specific agonist and antagonist peptides and for the generation of monoclonal antibodies (Mab). GRP interacts with a receptor coupled to a G protein and the signalling process leads to the activation of phospholipase C and kinases, and the mobilization of calcium. GRP promotes the proliferation of foetal and adult bronchial epithelium and of small cell lung cancer (SCLC) cells. GRP is also an autocrine growth factor for some SCLC cell lines. The growth of these lines is reduced in vitro and in vivo by MAb and specific antagonists. Hyperplasia of GRP producing cells has been shown in various lung diseases in adults and children. Pharmacological data on GRP suggest that its antagonists could be used in the treatment of SCLC (in addition to chemotherapy) and of interstitial lung disease. The cloning of the GRP receptor should facilitate the design of specific and potent antagonists of the peptide.

Adult↗
PubMed1991

Estradiol and EGF requirements for cell-cycle progression of normal human mammary epithelial cells in culture.

The purpose of our study was to show the feasibility of accurately investigating the factors likely to control cell proliferation of normal human mammary epithelial (HME) cells, using a scanning cytometric method. The methodology was previously developed with the SAMBA 200 cell image processor to characterize in situ the cell-cycle phases of HME cells. Since various compartments constitute the mammary epithelium, cells obtained after reduction mammoplasty were cultured in a medium with a low calcium content (0.06 mM) to provide proliferating normal HME cells while maintaining their differentiation characteristics. Estradiol and EGF requirements for cell-cycle phase progression of these cells were examined. Then, we showed that 2 normal HME cell cultures displaying different phenotypic characteristics may differently progress through the cell cycle under the same hormonally defined conditions. Cell-cycle progression of the epithelial cells presenting luminal phenotype was induced only by sequential stimulation/pretreatment with estradiol followed by EGF treatment; this progression was enhanced when estradiol was maintained during EGF treatment. This definite order demonstrated that estradiol could have a permissive effect on EGF mitogenic activity. In contrast, epithelial cells likely to be localized in the basal position in the mammary gland showed the same proliferating activity whatever the estradiol and EGF treatment. These cells progressed profusely in cell cycle, independent of exogenous estradiol and EGF contribution, but they remained sensitive to estradiol regarding EGF-receptor detection. Our results suggest autonomous proliferation of these cells through an autocrine pathway, in the absence of negative regulators.

Adult↗
PubMed1991

Gs alpha availability to cholera toxin-catalysed ADP-ribosylation is decreased in membranes of retinoic acid-treated leukemic cell lines HL-60 and THP-1. A posttranslational effect.

Retinoic acid (RA) induces HL-60 and THP-1 leukemic cell lines to differentiate into granulocyte-like and monocyte-like cells. Limited data are available concerning the effects of RA on components of the cyclic AMP pathway in human myeloid leukemic cells. We showed previously a decrease in adenylate cyclase activity in the presence of histamine, prostaglandin E1 and forskolin in RA-treated HL-60 cells as compared to untreated cells. We examined the elements of the signal transduction pathway utilized by RA in the human myeloid cell line HL-60 and the human monocytic cell line THP-1. We therefore studied the effect of RA on the activity of the stimulatory G-protein (Gs). We demonstrate that addition of RA to two human myeloid leukemia cell lines, HL-60 and THP-1, does not induce a reduction of the 2 subunit of Gs (Gs alpha) RNA or Gs alpha protein in the plasma membrane but leads to a rapid decrease in the cholera toxin (CTX)-catalysed ADP-ribosylation of Gs alpha. In addition, this effect seems to be specific to RA, since there was no modification in Gs alpha ADP-ribosylation in the membranes of cells treated with dimethyl sulfoxide (DMSO), another inducer of differentiation in HL-60 cells.

Adenosine Diphosphate↗
PubMed1991

Histological and urinary reactivity of monoclonal antibody 1BE12 in bladder carcinoma. Purification of the antigen from urine.

We have previously reported the production of monoclonal antibody (MAb) 1BE12, which recognizes a glycoprotein in breast-cancer cells. In the present work, 1BE12 reactivity was tested by immunohistochemistry in bladder carcinoma (92 cases) and in non-tumoral bladder samples (15 cases). In 71% of bladder tumors, more than 30% of cells were intensely stained by 1BE12. The percentage of reactive cells was higher in cancers invading the muscle than in more superficial tumors (p = 0.039). In non-tumoral bladder, immuno-staining, when present, was usually confined to the superficial layers with a low number of cells stained (less than 30%) in 13/15 cases. Slot blots, performed on urine samples from 43 bladder-cancer patients and 21 healthy controls, were quantified by densitometry scanning. We found higher optical density (OD) values in urine from muscle-invasive-cancer patients than in urine from more superficial tumors and healthy controls, with a significantly different distribution (p = 0.005). The urinary antigen was detected by immunoblotting with 1BE12 as high-molecular-weight species (greater than 150 kDa). The reactive glycoprotein could thus be purified by immunoaffinity and FPLC filtration from the perchloric-acid-soluble fraction of urine from patients with invasive bladder carcinoma. The availability of purified antigen will allow us to quantitate our assay, in order to evaluate its potential use as a prognostic indicator in bladder-cancer patients.

Antibodies, Monoclonal↗
PubMed1991

Immortalization of human adult normal prostatic epithelial cells by liposomes containing large T-SV40 gene.

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.

Acid Phosphatase↗
PubMed1991

Two monoclonal antibodies identify antigens preferentially expressed on normal human breast cells versus breast cancer cells.

In order to obtain antibodies with specificity toward normal mammary epithelial antigenic determinants, we immunized BALB/c mice with normal milk cells and screened the hybridomas against an undifferentiated breast cancer cell line H466B, peripheral blood lymphocytes and normal fibroblasts. Two hybridomas were generated, which produced BA6 (IgG1) and CA4 (IgM) monoclonal antibodies (MAbs). These MAbs did not react with 5 breast cancer cell lines. In cryostat sections of normal human breast tissue, BA6 was reactive with 6/6 and CA4 was reactive with 12/13 specimens both showing an apical staining of epithelial cells. Conversely staining of malignant cells in breast cancer biopsies was observed in 4/33 specimens with BA6 and in 4/19 specimens with CA4. Computerized image analysis (SAMBA) of immunostained sections showed homogeneous distribution of staining, with a high percentage of stained cell surfaces in normal breast (mean percentages of positive surfaces : BA6 : 75% and CA4 : 82%) while, in malignant samples, staining was heterogeneous, with a mean percentage of positive surface of 25% for BA6 and 12% for CA4. Both MAbs reacted strongly with human milk fat globule membranes (HMFGM) and skimmed milk. FPLC size exclusion chromatography of skimmed milk showed that CA4 and BA6 reactive materials eluted in distinct peaks in high molecular weight ranges. Electrophoretic separation of HMFGM followed by CA4 staining detected a high molecular weight reactive band (Mr 380-600 kDa). CA4 and BA6 reactivity was reduced by protease treatment of the antigen but was not affected by neuraminidase digestion, by methanol extraction or by Na-metaperiodate oxidation. After perchloric acid treatment of HMFGM, BA6 activity was lost while the CA4 activity was found in the soluble fraction. The results reported suggest that the two MAbs identify two distinct novel epitopes of normal breast cells.

Animals↗
PubMed1991

Increased intraplatelet levels of platelet-derived growth factor and transforming growth factor-beta in patients with myelofibrosis with myeloid metaplasia.

Platelet-derived growth factor (PDGF) is thought to play some role in the genesis of fibrosis associated with myeloproliferative disorders. In addition, transforming growth factor-beta (TGF-beta) has been confirmed to promote fibrotic process. Both PDGF and TGF-beta have been shown to cooperate with epidermal growth factor (EGF) in regulating the growth of human marrow fibroblasts. All three are contained in platelet alpha-granules. We report the results of a study in patients with myelofibrosis with myeloid metaplasia (MMM). We evaluated PDGF, TGF-beta and EGF-like activities in circulating platelets from patients compared to healthy subjects. In contrast to EGF-like intraplatelet levels which were similar in patients and in normal donors (1-4 ng/10(9) platelets), we found constantly higher values for both PDGF and TGF-beta in MMM patients. In both radioimmunoassay (RIA) and assay for mitogenic activity on human bone marrow fibroblasts, PDGF levels were increased on the average 2-3.5-fold over the levels found in normal donors (P less than 0.01 and P less than 0.001, respectively). PDGF serum levels in patients were consistent with those found in platelets. In platelet-poor plasma (PPP), PDGF concentrations were undetectable or congruent to 2 ng/ml in patients and in control donors as well. The total TGF-beta activity in platelet lysates, determined using a competitive radioreceptor binding assay on Swiss 3T3 mouse cells and an inhibition growth assay on CCL64 cells, was found 2-3-fold increased in patients with MMM as compared to control subjects (P less than 0.003). These results emphasize that, not only PDGF, but also TGF-beta are implicated in the myelofibrosis with myeloid metaplasia.

Adult↗
PubMed1991

Interferon-gamma in vivo reverses the increased platelet levels of platelet-derived growth factor and transforming growth factor-beta in patients with myelofibrosis with myeloid metaplasia.

We previously reported high platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) levels in platelets from patients with myelofibrosis with myeloid metaplasia. We report here the effects of in vivo administration of recombinant human interferon-gamma on these growth factor levels. Of six patients who entered this study in our institution, four completed the 6-month therapy trial with interferon-gamma PDGF and TGF-beta levels in platelets were evaluated before and after treatment. PDGF levels were determined using a radioimmunoassay and TGF-beta activity was measured using an inhibition growth assay on CC164 cells. In these four patients, IFN-gamma therapy led to a reduction of the increased PDGF and TGF-beta levels in platelets towards the normal range. PDGF and TGF-beta values decreased by 25-45% and 32-46%, respectively. An acute deterioration was observed in one patient 5 months after the end of IFN-gamma therapy. In this patient, the deterioration of his clinical state correlated with an increase in intraplatelet PDGF and TGF-beta levels.

Adult↗
PubMed1991

P-glycoprotein expression and in vitro reversion of doxorubicin resistance by verapamil in clinical specimens from acute leukaemia and myeloma.

The expression of the P-glycoprotein which is associated with the development of multidrug resistance in various cell lines was investigated in 87 fresh acute leukaemia and multiple myeloma samples using the specific mouse monoclonal antibody MRK16 in an indirect immunofluorescence assay. Considering a 10% positive cell cut-off value, a heterogeneous expression of P-glycoprotein was observed in 5/22 (22.7%) de novo acute leukaemias, 7/22 (31.8%) relapse or secondary acute leukaemias, 14/27 (51.8%) acute transformation of myeloproliferative or myelodysplastic syndromes and 5/16 (31.2%) multiple myelomas. This expression was not associated with specific cytogenetic abnormalities, especially alterations of chromosome 7q. Verapamil, a calcium channel blocker, has been demonstrated to circumvent the multidrug resistance in cell lines, possibly by interfering with P-glycoprotein function. Using the microculture tetrazolium assay, verapamil was demonstrated to increase the sensitivity of fresh leukaemic or myeloma cells to doxorubicin in 19/43 (43.1%) samples. The doxorubicin IC50 level and the capacity of verapamil to increase the sensitivity of blast cells to doxorubicin in vitro did not correlate with the expression of P-glycoprotein. We conclude that high non-cytotoxic concentrations of verapamil were able to increase the in vitro doxorubicin sensitivity of fresh acute leukaemia and myeloma cells without detectable expression of the P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

[Lipid profiles of breast cancer cell lines: proton nuclear magnetic resonance spectroscopy].

Nuclear magnetic resonance spectroscopy was performed on breast cancer cell lines MCF-7, MDA-MB231 and T47D. Proton spectra showed discrepancies of lipid quantity in the different lines. The high resolution lines of lipids were not as intense in the membrane preparations. These results show the potential of NMR spectroscopy to study the involvement of membrane lipids in proliferation, metastasis or drug resistance process.

Adenocarcinoma↗
PubMed1990

Characterization and distribution in human tissues of a glycoproteic antigen defined by monoclonal antibody 1BE12 raised against the human breast cancer cell line T47D.

Spleen cells from inbred Biozzi mice, immunized against the human breast cancer cell line T47D, were fused with murine myeloma SP2O cells to generate monoclonal antibodies. One of these, 1BE12, of IgM isotype, reacted with five of six human breast tumor cell lines, while no binding was detectable with normal lymphocytes, RBC, or fibroblasts. The antigen recognized by monoclonal antibody 1BE12 was localized on the surface of T47D and MCF7 cells and was detected in cell-free supernatants of cultures. The antigen was found also on the surface of milk secretory cells. Immunohistochemical staining of frozen and paraffin-embedded sections of human tissues showed apical polarized reactivity in normal breast glands, while in all breast cancers staining was either cytoplasmic or membranous and heterogeneously distributed. Immunostaining was also observed in some other normal epithelia, including salivary gland, gastroduodenal mucosa, exocrine pancreas, and cervix. The antigen was not detectable in secretory endometrium, whereas proliferative endometrium was strongly stained. Colon carcinoma, and cancers of the bladder and endometrium were strongly reactive. No staining was detected in melanoma, lymphoma, mesothelioma, non-small cell lung carcinoma, and thyroid, renal, and ovarian carcinomas. Lectin absorption of MCF7 membrane extracts reduced 1BE12 binding. A large reduction in 1BE12 reactivity was observed after digestion of T47D and MCF7 membrane extracts with proteases. Treatment with sodium periodate resulted in complete loss of antigenicity, while neuraminidase treatment did not affect 1BE12 binding. These findings suggest that the 1BE12 epitope is expressed on the carbohydrate moiety of a glycoprotein and does not contain sialic acid. Immunoblotting of the perchloric acid-soluble fraction of MCF7 membrane extracts after electrophoresis in 1% agarose detected the antigen as a high molecular weight species (Mr greater than 900,000). The antigen was purified by perchloric acid extraction of MCF7 membrane preparations followed by affinity chromatography on 1BE12 antibody coupled to Sepharose-4B and gel exclusion fast protein liquid chromatography. No reactivity of the purified material was found with monoclonal antibodies directed against human milk fat globule membrane-associated mucins HMFG1 and DF3.

Antibodies, Monoclonal↗